RESUMO
Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.
Assuntos
Glutationa , Ratos , Transcobalaminas , Vitamina B 12 , Animais , Cristalografia por Raios X , Glutationa/metabolismo , Glutationa/análogos & derivados , Glutationa/química , Ligação Proteica , Transcobalaminas/metabolismo , Transcobalaminas/química , Vitamina B 12/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMO
This review describes the adaptation of the plant virus-based transient expression system, magnICON(®) for the at-scale manufacturing of pharmaceutical proteins. The system utilizes so-called "deconstructed" viral vectors that rely on Agrobacterium-mediated systemic delivery into the plant cells for recombinant protein production. The system is also suitable for production of hetero-oligomeric proteins like immunoglobulins. By taking advantage of well established R&D tools for optimizing the expression of protein of interest using this system, product concepts can reach the manufacturing stage in highly competitive time periods. At the manufacturing stage, the system offers many remarkable features including rapid production cycles, high product yield, virtually unlimited scale-up potential, and flexibility for different manufacturing schemes. The magnICON system has been successfully adaptated to very different logistical manufacturing formats: (1) speedy production of multiple small batches of individualized pharmaceuticals proteins (e.g. antigens comprising individualized vaccines to treat NonHodgkin's Lymphoma patients) and (2) large-scale production of other pharmaceutical proteins such as therapeutic antibodies. General descriptions of the prototype GMP-compliant manufacturing processes and facilities for the product formats that are in preclinical and clinical testing are provided.
Assuntos
Anticorpos Monoclonais/biossíntese , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Receptores CCR5/imunologia , Proteínas Recombinantes/biossíntese , HumanosRESUMO
Antibody-based products are not widely available to address many global health challenges due to high costs, limited manufacturing capacity, and long manufacturing lead times. There are now tremendous opportunities to address these industrialization challenges as a result of revolutionary advances in plant virus-based transient expression. This review focuses on some antibody-based products that are in preclinical and clinical development, and have scaled up manufacturing and purification (mg of purified mAb/kg of biomass). Plant virus-based antibody products provide lower upfront cost, shorter time to clinical and market supply, and lower cost of goods (COGs). Further, some plant virus-based mAbs may provide improvements in pharmacokinetics, safety and efficacy.
Assuntos
Anticorpos Monoclonais/genética , Vírus de Plantas/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Vacinas contra Ebola/uso terapêutico , Humanos , Idiótipos de Imunoglobulinas/uso terapêuticoRESUMO
Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.
Assuntos
Butirilcolinesterase/metabolismo , Nicotiana/metabolismo , Butirilcolinesterase/genética , Butirilcolinesterase/isolamento & purificação , Vetores Genéticos/metabolismo , Glicosilação , Humanos , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes/metabolismo , Nicotiana/genéticaRESUMO
The development of an efficacious vaccine against norovirus is of paramount importance given its potential to reduce the global burden of norovirus-associated morbidity and mortality. Here, we report a detailed immunological analysis of a phase I, double-blind, placebo-controlled clinical trial performed on 60 healthy adults, ages 18 to 40. Total serum immunoglobulin and serum IgA against vaccine strains and cross-reactive serum IgG against non-vaccine strains were measured by enzyme immunoassays, whereas cell-mediated immune responses were quantified using intracellular cytokine staining by flow cytometry. A significant increase in humoral and cellular responses, e.g., IgA and CD4+ polypositive T cells, was triggered by the GI.4 Chiba 407 (1987) and GII.4 Aomori 2 (2006) VLP-based norovirus vaccine candidate rNV-2v, which is formulated without adjuvant. No booster effect was observed after the second administration in the pre-exposed adult study population. Furthermore, a cross-reactive immune response was elicited, as shown by IgG titers against GI.3 (2002), GII.2 OC08154 (2008), GII.4 (1999), GII.4 Sydney (2012), GII.4 Washington (2018), GII.6 Maryland (2018), and GII.17 Kawasaki 308 (2015). Due to viral infection via mucosal gut tissue and the high variety of potentially relevant norovirus strains, a focus should be on IgA and cross-protective humoral and cell-mediated responses in the development of a broadly protective, multi-valent norovirus vaccine. Clinical trial registration: https://clinicaltrials.gov, identifier NCT05508178. EudraCT number: 2019-003226-25.
Assuntos
Adjuvantes Imunológicos , Norovirus , Adulto , Humanos , Vacinas Combinadas , Adjuvantes Farmacêuticos , Imunoglobulina A , Imunoglobulina GRESUMO
Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide. A safe and effective vaccine that prevents NoV infection or minimizes NoV disease burden is needed, especially for children and the elderly who are particularly susceptible to NoV disease. A plant-based expression system (magnICON®) was used to manufacture two different virus-like particle (VLP) immunogens derived from human NoV genogroups I and II, genotype 4 (GI.4 and GII.4), which were subsequently blended 1:1 (w/w) into a bivalent vaccine composition (rNV-2v). Here, we report on the safety and immunogenicity of rNV-2v from one pilot and two GLP-compliant toxicity studies in New Zealand White rabbits administered the vaccine subcutaneously (SC) or intramuscularly (IM). Strong genogroup-specific immune responses were induced by vaccination without adjuvant at various doses (200 to 400 µg VLP/administration) and administration schedules (Days 1 and 7; or Days 1, 15 and 29). The results showed sporadic local irritation at the injection site, which resolved over time, and was non-adverse and consistent with expected reactogenicity. There were no signs of systemic toxicity related to vaccine administration relative to vehicle-treated controls with respect to clinical chemistry, haematology, organ weights, macroscopic examinations, or histopathology. In a 3-administration regimen (n + 1 the clinical regimen), the NOAEL for rNV-2v via the SC or IM route was initially determined to be 200 µg. An improved GI.4 VLP variant mixed 1:1 (w/w) with the wild-type GII.4 VLP was subsequently evaluated via the IM route at a higher dose in the same 3-administration model, and the NOAEL was raised to 300 µg. Serology performed in samples of both toxicity studies showed significant and substantial anti-VLP-specific antibody titers for rNV-2v vaccines administered via the IM or SC route, as well as relevant NoV blocking antibody responses. These results support initiation of clinical development of the plant-made NoV vaccine.
Assuntos
Infecções por Caliciviridae , Norovirus , Vacinas de Partículas Semelhantes a Vírus , Vacinas Virais , Animais , Anticorpos Antivirais , Modelos Animais , CoelhosRESUMO
Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide and a safe and effective vaccine is needed. Here, a phase I, double-blind, placebo-controlled clinical trial was performed in 60 healthy adults, 18 to 40 years old. Safety (primary objective) and immunogenicity (secondary and exploratory objectives) of a bivalent (GI.4 and GII.4), plant-produced, virus-like particle (VLP), NoV vaccine candidate formulation were investigated at two dose levels (50 µg + 50 µg and 150 µg + 150 µg) without adjuvant. Overall, 13 subjects (65.0%) in the 50 µg group, 16 subjects (80.0%) in the 150 µg group, and 14 subjects (70.0%) in the placebo group reported at least 1 solicited local or general symptom during the 7-day post-vaccination periods following each dose. Severe solicited adverse events (AEs) were rare (2 events in the 50 µg group). A total of 8 subjects (40.0%) in each group reported at least one unsolicited AE during the 28-day post-vaccination periods. Immunogenicity was assessed on days 1, 8, 29, 57, 183 and 365. All subjects were pre-exposed to norovirus as indicated by baseline levels of the different immunological parameters examined. Vaccine-specific humoral and cellular immune responses increased after the first dose but did not rise further after the second vaccination. Increased GI.4- and GII.4-specific IgG titers persisted until day 365. The vaccine elicited cross-reactive IgG antibodies against non-vaccine NoV VLPs, which was more pronounced for NoV strains of the same genotype as the GII.4 vaccine strain than for non-vaccine genotypes. Significant blocking anti-GI.4 and anti-GII.4 VLP titers were triggered in both dose groups. Lymphoproliferation assays revealed strong cell-mediated immune responses that persisted until day 365. In conclusion, both dose levels were safe and well-tolerated, and no higher incidence of AEs was observed in the higher dose group. The data show that a single dose of the vaccine formulated at 50 µg of each VLP is sufficient to reach a peak immune response after 8 to 28 days. The results of this Phase I study warrant further evaluation of the non-adjuvanted vaccine candidate. Clinical trial registration: https://clinicaltrials.gov/ct2/show/record/NCT05508178, identifier (NCT05508178).
Assuntos
Infecções por Caliciviridae , Gastroenterite , Norovirus , Vacinas Virais , Adulto , Humanos , Adolescente , Adulto Jovem , Imunoglobulina G , Adjuvantes ImunológicosRESUMO
Highly pathogenic avian influenza (HPAI) is a striking disease in susceptible poultry, which leads to severe economic losses. Inactivated vaccines are the most widely used vaccines in avian influenza virus (AIV) vaccination programs. However, these vaccines interfere with the serological detection of wild-type AIV infections in immunized populations. The use of vaccines that allow differentiation between infected and vaccinated animals (DIVA strategy) would stop current stamping-out policies. Therefore, novel vaccination strategies are needed to allow improved protection of animals and humans against HPAI virus (HPAIV) infection. The presented study analyzed for the first time the immunogenic capacity of plant-expressed full-length hemagglutinin (rHA0) of HPAIV H5N1 in several vaccine formulations within the highly relevant host species chicken. We were able to express plant-expressed rHA0 at high levels and could show that, when administered with potent adjuvants, it is highly immunogenic and can fully protect chicken against lethal challenge infection. Real-time reverse transcription (RT)-PCR and serological tests demonstrated only marginally increased virus replication in animals vaccinated with plant-derived rHA0 compared to animals immunized with an inactivated reference vaccine. In addition, the use of plant-expressed rHA0 also allowed an easy serological differentiation of vaccinated from AIV-infected animals based on antibodies against the influenza virus NP protein.
Assuntos
Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Nicotiana/genética , Animais , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Imunização , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/fisiologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Nicotiana/metabolismoRESUMO
A two-component hybrid seed system has been developed that is broadly applicable and provides for effective generation and maintenance of the male-sterile parent, hybrid seed production and full restoration of fertility in the hybrid seed. The technology is based on the functional interaction of two loci that are inserted in the same position on two homologous chromosomes, and thus are 'linked in repulsion', and that jointly code for male sterility and herbicide resistance, both traits being expressed in heterozygous plants only. The localization to the same locus on a chromosome is achieved by the genetic transformation of plants with a construct containing both genetic elements (loci), and subsequent derivatization from the primary pro-locus of the two precursor lines using site-specific deletions. The functional interaction of the two loci is achieved through intein-based trans-splicing of two pairs of complementary protein fragments that provide for male sterility and herbicide resistance. Unlike the hybrid seed systems that are currently in use, the technology relies on the genetic modification of just one parent, and is therefore much simpler to develop and use. Arabidopsis has been used for the proof of principle presented here, but the essential elements of the technology are generic and have been shown to work in many crop species.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Hibridização Genética , Sementes/genética , Sementes/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias , Fertilidade/genética , Engenharia Genética , Genótipo , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Fenótipo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Ribonucleases/genética , Nicotiana/genéticaRESUMO
The use of plant viral vectors for the transient expression of heterologous proteins offers a useful tool for the large-scale production of proteins of industrial importance, such as antibodies and vaccine antigens. In recent years, advances have been made both in the development of first-generation vectors (that employ the 'full virus') and second-generation ('deconstructed virus') vectors. For example, vectors based around the 'full virus' strategy can now be used to express long polypeptides (at least 140 amino acids long) as fusions to the coat protein. In addition, a new generation of vectors was engineered to have a reactogenic amino acid exposed on the surface of the virus, allowing easy chemical conjugation of (separately produced) proteins to the viral surface. This approach is being used to develop new vaccines in the form of antigens coupled to a plant viral surface. Prototypes of industrial processes that require high-yield production, rapid scale-up, and fast manufacturing have been recently developed using the 'deconstructed virus' approach (magnifection). This process, which relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons to plant cells, has been shown to work with numerous proteins, including full immunoglobulin G antibodies. Other advances in this area have looked at the development of inducible viral systems and the use of viral vectors to produce nanoscale materials for modular assembly.
Assuntos
Vetores Genéticos/genética , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Vírus/genéticaRESUMO
Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium-mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection). This process, called "magnifection", can be performed on a large scale and with different plant species. This technology combines advantages of three biological systems (the transfection efficiency of A. tumefaciens, the high expression yield obtained with viral vectors, and the post-translational capabilities of a plant), does not require genetic modification of plants and is faster than other existing methods.
Assuntos
Agrobacterium tumefaciens/fisiologia , Vetores Genéticos , Nicotiana/genética , Nicotiana/metabolismo , RNA Viral/genética , Vírus do Mosaico do Tabaco/genética , Transfecção/métodos , Beta vulgaris/genética , Beta vulgaris/metabolismo , Expressão Gênica , Engenharia Genética/métodos , Genoma Viral , Plantas Geneticamente Modificadas , Replicação ViralRESUMO
Plant viral vectors are being successfully developed and exploited for the industrial-scale expression of heterologous proteins and as a research tool for studies of gene expression. The initial engineering strategy (the 'full virus' vector strategy) aimed to design a vector that was essentially a wildtype virus, which was modified to carry and express a heterologous sequence that encoded a gene of interest. The new emerging trend (the 'deconstructed virus' vector strategy) reflects an ideology that recognises the inherent limitations of the viral process. It attempts to 'deconstruct' the virus, by eliminating functions that are limiting or undesired, and to rebuild it, either by delegating the missing necessary functions to the host (which is genetically modified to provide those functions) or by replacing them with analogous functions that are not derived from a virus.
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/genética , Expressão Gênica , Plantas Geneticamente Modificadas/virologiaRESUMO
Co-agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, co-expression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Nicotiana/enzimologia , Interferência de RNA , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Precursores Enzimáticos , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , MicroRNAs/biossíntese , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Proteínas de Plantas/metabolismo , Transporte Proteico , Estabilidade de RNA/genética , RNA Interferente Pequeno/biossíntese , RNA Viral/metabolismo , Rhizobium/genética , Vírus do Mosaico do Tabaco/fisiologia , TransgenesRESUMO
Passive antibody therapy for cancer is an effective but costly treatment modality. Induction of therapeutically potent anticancer antibodies by active vaccination is an attractive alternative but has proven challenging in cancer due to tolerogenic pressure in patients. Here, we used the clinically relevant cancer target Her2, known to be susceptible to targeting by antibody therapy, to demonstrate how potent antibody can be induced by vaccination. A novel 44kD Her2 protein fragment was generated and found to be highly effective at inducing anti-Her2 antibody including trastuzumab-like reactivities. In the tolerant and spontaneous BALB-neuT mouse model of metastatic breast cancer this Her2-targeting vaccine was only effective if the fragment was conjugated to a foreign immunogenic carrier; Fragment C of tetanus toxin. Only the conjugate vaccine induced high affinity anti-Her2 antibody of multiple isotypes and suppressed tumor development. The magnitude of CD4(+) T-cell help and breadth of cytokines secreted by the CD4(+) T helper (Th) cells induced to the foreign antigen was critical. We used a highly efficient plant-based bio-manufacturing process for protein antigens, magnICON, for vaccine expression, to underpin feasibility of future clinical testing. Hence, our novel Her2-targeting conjugate vaccine combines preclinical efficacy with clinical deliverability, thus setting the scene for therapeutic testing.
RESUMO
We describe here a high-yield transient expression system for the production of human growth hormone (hGH, or somatotropin) in transfected Nicotiana benthamiana leaves. The system is based on a recently described plant virus-based modular expression vector [Gleba, Y., Marillonnet, S. and Klimyuk, V. (2004) Engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies. Curr. Opin. Plant Biol. 7, 182-188; Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V. and Gleba, Y. (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA, 101, 6852-6857], and represents a simple and fast alternative to stable transformation. By using various combinations of provector modules, hGH was produced in three compartments of the cell: the apoplast, the chloroplast and the cytosol. We found that targeting to the apoplast provided the highest amount of correctly processed and biologically active hGH, with a yield of up to 10% of total soluble protein or 1 mg per gram of fresh weight leaf biomass. These results indicate that the use of viral vectors for high-yield production of human therapeutic proteins in plants by transient expression provides an attractive alternative to production protocols using standard expression vectors in transgenic or transplastomic plants.
RESUMO
We report the first evaluation of plant-made conjugate vaccines for targeted treatment of B-cell follicular lymphoma (FL) in a Phase I safety and immunogenicity clinical study. Each recombinant personalized immunogen consisted of a tumor-derived, plant-produced idiotypic antibody (Ab) hybrid comprising the hypervariable regions of the tumor-associated light and heavy Ab chains, genetically grafted onto a common human IgG1 scaffold. Each immunogen was produced in Nicotiana benthamiana plants using twin magnICON vectors expressing the light and heavy chains of the idiotypic Ab. Each purified Ab was chemically linked to the carrier protein keyhole limpet hemocyanin (KLH) to form a conjugate vaccine. The vaccines were administered to FL patients over a series of ≥6 subcutaneous injections in conjunction with the adjuvant Leukine (GM-CSF). The 27 patients enrolled in the study had previously received non-anti-CD20 cytoreductive therapy followed by ≥4 months of immune recovery prior to first vaccination. Of 11 patients who became evaluable at study conclusion, 82% (9/11) displayed a vaccine-induced, idiotype-specific cellular and/or humoral immune response. No patients showed serious adverse events (SAE) related to vaccination. The fully scalable plant-based manufacturing process yields safe and immunogenic personalized FL vaccines that can be produced within weeks of obtaining patient biopsies.
Assuntos
Hemocianinas/imunologia , Linfoma Folicular/imunologia , Nicotiana/metabolismo , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologia , Adolescente , Adulto , Idoso , Demografia , Feminino , Hemocianinas/efeitos adversos , Humanos , Imunidade Celular , Imunidade Humoral , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Polissacarídeos/imunologia , Vacinação , Adulto JovemRESUMO
CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.Molecular Therapy - Nucleic Acids (2013) 2, e98; doi:10.1038/mtna.2013.26; published online 11 June 2013.