Clinical Safety and Immunogenicity of Tumor-Targeted, Plant-Made Id-KLH Conjugate Vaccines for Follicular Lymphoma.

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.

CD28 aptamers as powerful immune response modulators.

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.

Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes.

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.

Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call 'Golden Gate shuffling', is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.

A one pot, one step, precision cloning method with high throughput capability.

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants.

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.

High-yield production of authentic human growth hormone using a plant virus-based expression system.

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.

In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3-4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana.

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.