RESUMEN
Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to â¼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.
Asunto(s)
Silenciador del Gen , Interferencia de ARN , ARN Mensajero , Animales , Humanos , Ratones , Ratones Transgénicos , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
T cell Receptor (TCR) Fusion Construct (TRuC®) T cells harness all signaling subunits of the TCR to activate T cells and eliminate tumor cells, with minimal release of cytokines. While adoptive cell therapy with chimeric antigen receptor (CAR)-T cells has shown unprecedented clinical efficacy against B-cell malignancies, monotherapy with CAR-T cells has suboptimal clinical efficacy against solid tumors, probably because of the artificial signaling properties of the CAR. TRuC-T cells may address the suboptimal efficacy of existing CAR-T therapies for solid tumors. Here, we report that mesothelin (MSLN)-specific TRuC-T cells (referred to as TC-210 T cells) potently kill MSLN+ tumor cells in vitro and efficiently eradicate MSLN+ mesothelioma, lung, and ovarian cancers in xenograft mouse tumor models. When benchmarked against MSLN-targeted BBζ CAR-T cells (MSLN-BBζ CAR-T cells), TC-210 T cells show an overall comparable level of efficacy; however, TC-210 T cells consistently show faster tumor rejection kinetics that are associated with earlier intratumoral accumulation and earlier signs of activation. Furthermore, in vitro and ex vivo metabolic profiling suggests TC-210 T cells have lower glycolytic activity and higher mitochondrial metabolism than MSLN-BBζ CAR-T cells. These data highlight TC-210 T cells as a promising cell therapy for treating MSLN-expressing cancers. The differentiated profile from CAR-T cells may translate into better efficacy and safety of TRuC-T cells for solid tumors.
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Mesotelioma Maligno , Neoplasias Ováricas , Humanos , Animales , Ratones , Femenino , Linfocitos T , Mesotelina , Receptores de Antígenos de Linfocitos T , Modelos Animales de EnfermedadRESUMEN
The development of an aptamer-based therapeutic has rapidly progressed following the first two reports in the 1990s, underscoring the advantages of aptamer drugs associated with their unique binding properties. In 2004, the US Food and Drug Administration (FDA) approved the first therapeutic aptamer for the treatment of neovascular age-related macular degeneration, Macugen developed by NeXstar. Since then, eleven aptamers have successfully entered clinical trials for various therapeutic indications. Despite some of the pre-clinical and clinical successes of aptamers as therapeutics, no aptamer has been approved by the FDA for the treatment of cancer. This review highlights the most recent and cutting-edge approaches in the development of aptamers for the treatment of cancer types most refractory to conventional therapies. Herein, we will review (1) the development of aptamers to enhance anti-cancer immunity and as delivery tools for inducing the expression of immunogenic neoantigens; (2) the development of the most promising therapeutic aptamers designed to target the hard-to-treat cancers such as brain tumors; and (3) the development of "carrier" aptamers able to target and penetrate tumors and metastasis, delivering RNA therapeutics to the cytosol and nucleus.
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Aptámeros de Nucleótidos/uso terapéutico , Neoplasias/tratamiento farmacológico , Aptámeros de Nucleótidos/inmunología , Portadores de Fármacos , Desarrollo de Medicamentos , Humanos , Neoplasias/inmunología , Escape del Tumor/efectos de los fármacosRESUMEN
Vaccination of patients against neoantigens expressed in concurrent tumors, recurrent tumors, or tumors developing in individuals at risk of cancer is posing major challenges in terms of which antigens to target and is limited to patients expressing neoantigens in their tumors. Here, we describe a vaccination strategy against antigens that were induced in tumor cells by downregulation of the peptide transporter associated with antigen processing (TAP). Vaccination against TAP downregulation-induced antigens was more effective than vaccination against mutation-derived neoantigens, was devoid of measurable toxicity, and inhibited the growth of concurrent and future tumors in models of recurrence and premalignant disease. Human CD8+ T cells stimulated with TAPlow dendritic cells elicited a polyclonal T-cell response that recognized tumor cells with experimentally reduced TAP expression. Vaccination against TAP downregulation-induced antigens overcomes the main limitations of vaccinating against mostly unique tumor-resident neoantigens and could represent a simpler vaccination strategy that will be applicable to most patients with cancer.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Recurrencia Local de Neoplasia/terapia , Neoplasias/terapia , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/inmunología , Animales , Presentación de Antígeno/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/inmunología , Neoplasias/inmunología , ARN Interferente Pequeño/genéticaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Antígenos de Neoplasias/inmunología , Inmunoterapia , Neoplasias/inmunología , Neoplasias/prevención & control , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antígenos de Neoplasias/genética , Aptámeros de Nucleótidos , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Vacunas contra el Cáncer , Línea Celular Tumoral , Regulación hacia Abajo , Epítopos/inmunología , Femenino , Humanos , Inmunización , Inmunogenicidad Vacunal , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Células 3T3 NIH , Neoplasias Experimentales , Oligodesoxirribonucleótidos , Fosfoproteínas , Receptor de Muerte Celular Programada 1/inmunología , ARN Interferente Pequeño/administración & dosificación , Proteínas de Unión al ARN , Bazo/inmunología , Bazo/patología , Vacunación , NucleolinaRESUMEN
Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.
Asunto(s)
Neoplasias Encefálicas/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Glioblastoma/inmunología , Inmunidad Innata , Macrófagos/inmunología , Proteínas de Neoplasias/inmunología , Osteopontina/inmunología , Animales , Aptámeros de Nucleótidos/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/genética , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Osteopontina/genética , Receptores de Vitronectina/genética , Receptores de Vitronectina/inmunologíaRESUMEN
Uptake of tumor antigens by tumor-infiltrating dendritic cells is limiting step in the induction of tumor immunity, which can be mediated through Fc receptor (FcR) triggering by antibody-coated tumor cells. Here we describe an approach to potentiate tumor immunity whereby hapten-specific polyclonal antibodies are recruited to tumors by coating tumor cells with the hapten. Vaccination of mice against dinitrophenol (DNP) followed by systemic administration of DNP targeted to tumors by conjugation to a VEGF or osteopontin aptamer elicits potent FcR dependent, T cell mediated, antitumor immunity. Recruitment of αGal-specific antibodies, the most abundant naturally occurring antibodies in human serum, inhibits tumor growth in mice treated with a VEGF aptamer-αGal hapten conjugate, and recruits antibodies from human serum to human tumor biopsies of distinct origin. Thus, treatment with αGal hapten conjugated to broad-spectrum tumor targeting ligands could enhance the susceptibility of a broad range of tumors to immune elimination.
Asunto(s)
Anticuerpos/metabolismo , Haptenos/metabolismo , Animales , Dinitrofenoles/inmunología , Humanos , Inmunohistoquímica , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Osteopontina/metabolismo , Receptores Fc/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
TGFß secreted by tumor cells and/or tumor infiltrating stromal cells is a key mediator of tumor growth and immune suppression at the tumor site. Nonetheless, clinical trials in cancer patients targeting the TGFß pathway exhibited at best a modest therapeutic benefit. A likely reason, a common limitation of many cancer drugs, is that the physiologic roles of TGFß in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFß may be sufficient to inhibit tumor growth. Here we describe an approach to render vaccine-activated CD8+ T cells transiently resistant to TGFß inhibition using an siRNA against Smad4 to inhibit a key step in the canonical TGFß signaling pathway. The siRNA was targeted to vaccine activated CD8+ T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand (4-1BB-Smad4 conjugate). In vitro the 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFß inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFß to tumor-specific T cells will not interfere with its multiple physiologic roles and hence reduce the risk of toxicity.
RESUMEN
Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents. Cancer Res; 77(6); 1310-21. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Aptámeros de Nucleótidos/farmacología , Rayos gamma/efectos adversos , Terapia Molecular Dirigida , Neoplasias Experimentales/prevención & control , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/etiología , Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de la radiación , Células Tumorales Cultivadas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Aptámeros de Nucleótidos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de Señal , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular , Supervivencia Celular/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica , Memoria Inmunológica/inmunología , Inmunoterapia , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Neoplasias/terapia , Fenotipo , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
PURPOSE: To report a novel strategy using oligonucleotide aptamers to 4-1BB as an alternate method for costimulation, and show that combinatorial therapy with radiation improves the therapeutic ratio over equivalent monoclonal antibodies. METHODS AND MATERIALS: Subcutaneous 4T1 (mouse mammary carcinoma) tumors were established (approximately 100 mm(3)), and a radiation therapy (RT) dose/fractionation schedule that optimally synergizes with 4-1BB monoclonal antibody (mAb) was identified. Comparable tumor control and animal survival was observed when either 4-1BB antibody or aptamer were combined with RT using models of breast cancer and melanoma (4T1 and B16-F10). Off-target CD8(+) T-cell toxicity was evaluated by quantification of CD8(+) T cells in livers and spleens of treated animals. RESULTS: When combined with 4-1BB mAb, significant differences in tumor control were observed by varying RT dose and fractionation schedules. Optimal synergy between RT and 4-1BB mAb was observed at 5 Gy × 6. Testing 4-1BB mAb and aptamer independently using the optimal RT (5 Gy × 6 for 4T1/Balb/c and 12 Gy × 1 for B16/C57BL6J mouse models) revealed equivalent tumor control using 4-1BB aptamer and 4-1BB mAb. 4-1BB mAb, but not 4-1BB aptamer-treated animals, exhibited increased lymphocytic liver infiltrates and increased splenic and liver CD8(+) T cells. CONCLUSIONS: Radiation therapy synergizes with 4-1BB mAb, and this effect is dependent on RT dose and fractionation. Tumor control by 4-1BB aptamer is equivalent to 4-1BB mAb when combined with optimal RT dose, without eliciting off-target liver and spleen CD8(+) expansion. 4-1BB aptamer-based costimulation affords a comparable and less toxic strategy to augment RT-mediated tumor control.
Asunto(s)
Ligando 4-1BB/antagonistas & inhibidores , Aptámeros de Nucleótidos/administración & dosificación , Quimioradioterapia/métodos , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Ligando 4-1BB/inmunología , Animales , Línea Celular Tumoral , Terapia Combinada/métodos , Relación Dosis-Respuesta en la Radiación , Femenino , Factores Inmunológicos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Radioinmunoterapia/métodos , Dosificación Radioterapéutica , Resultado del TratamientoRESUMEN
High-grade gliomas are notoriously heterogeneous regarding antigen expression, effector responses, and immunosuppressive mechanisms. Therefore, combinational immune therapeutic approaches are more likely to impact a greater number of patients and result in longer, durable responses. We have previously demonstrated the monotherapeutic effects of miR-124, which inhibits the signal transducer and activator of transcription 3 (STAT3) immune suppressive pathway, and immune stimulatory 4-1BB aptamers against a variety of malignancies, including genetically engineered immune competent high-grade gliomas. To evaluate potential synergy, we tested an immune stimulatory aptamer together with microRNA-124 (miRNA-124), which blocks tumor-mediated immune suppression, and found survival to be markedly enhanced, including beyond that produced by monotherapy. The synergistic activity appeared to be not only secondary to enhanced CD3(+) cell numbers but also to reduced macrophage immune tumor trafficking, indicating that a greater therapeutic benefit can be achieved with approaches that both induce immune activation and inhibit tumor-mediated immune suppression within the central nervous system (CNS) tumors.
RESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Aptámeros de Nucleótidos/administración & dosificación , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/efectos adversos , Aptámeros de Nucleótidos/inmunología , Sistemas de Liberación de Medicamentos/métodos , Humanos , Terapia Molecular Dirigida/métodos , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Interferente Pequeño/inmunologíaRESUMEN
The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6â mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.
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Separación Celular/instrumentación , Separación Celular/métodos , Filtros Microporos , Células Neoplásicas Circulantes/patología , Animales , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Reproducibilidad de los ResultadosRESUMEN
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.