WO2014044892A1 - Method for the production of complex repertoires of recombinant molecules - Google Patents

Abstract

The invention relates to a method for the production of complex repertoires of recombinant molecules in a consistent and reproducible manner, comprising the transient generation of multi-transgenic plants that generate somatic mosaics induced by viral replicons that are mutually exclusive. The invention also relates to the multi-transgenic plant or a fragment of same obtained in this way, as well as to extracts or purified fractions thereof, which represent complex repertoires of recombinant molecules, preferably recombinant proteins selected from among: enzymes, immunoglobulins, membrane receptors, intracellular receptors, lectins, polyclonal antibodies, antidotes based on anti-serums, immunological serums for passive immunity, and intravenous immunoglobulins.

Description

 PRODUCTION METHOD OF COMPLEX MOLECLE REPERTORIES

 RECOM BINANTES

DESCRIPTION

SECTOR OF THE TECHNIQUE

The present invention falls within the "agricultural biotechnology" technology, being applicable in the production of recombinant molecule assemblies in the chemical, pharmaceutical, veterinary and agricultural sectors.

STATE OF THE PREVIOUS TECHNIQUE

Currently there is a growing demand for recombinant molecules, for use in different sectors such as pharmaceutical, veterinary, energy, etc. Many of these proteins require eukaryotic systems for their production. In most cases, the ultimate goal of the technique is the production of a single protein of homogeneous composition, such as a growth factor or a monoclonal antibody, and therefore most current systems are adapted to this end. . However, there are no methodologies that allow to produce complex repertoires of different proteins so that the final composition of the mixture is highly reproducible.

Some examples of complex sets of proteins that the industry produces and / or uses are polyclonal antibodies, hyperimmune immunoglobulins, immunological sera for passive immunity or antivenoms based on antisera. In all the cases cited, the set comprises, in whole or in part, the repertoire of amino acid sequences that make up the immune response of one or more individuals. The mammalian immune response bases much of its efficacy on the fact that it comprises a wide repertoire (polyclonal) of different immunoglobulins (antibodies). In this way different immunoglobulins recognize different regions of the pathogen / antigen / toxin ensuring greater adaptability and neutralization capacity. Similarly, polyclonal antibodies are more effective than monoclonal antibodies in many therapeutic applications, such as the neutralization of toxins and infectious agents, or diagnostic such as the detection of specimens of poorly defined composition (Bregenholt et al., 2006; Dunman and Nesin, 2003; Haurum, 2006; Koefoed et al., 2006; Sharon et al., 2000).

Despite the advantages inherent in polyclonality, the production of recombinantly complex protein repertoires has been poorly addressed due to its technical complexity. Proof of this is that currently commercial recombinant antibodies are monoclonal. In those applications where the use of complex repertoires is necessary, such as polyclonal antibodies for diagnosis, antivenoms based on antisera, intravenous immunoglobulins for the treatment of immunodeficiencies or hyperimmune immunoglobulins for passive immunization therapies, recourse to producing them in a traditional way (non-recombinant), by immunization and subsequent isolation and purification of polyclonal sera in animals (mouse, rabbit, goat or horse, among others) or humans.

Polyclonal antibodies, antivenoms based on antisera and other immunological products for passive immunity obtained from immunized animals present the following problems: (i) pose ethical problems in their manufacture; (ii) they have high variability between batches, due to differences in immunological response of different individuals (iii) they require a thorough de novo characterization in each batch (iv) they present risks of immunogenicity and inadvertent transmission of pathogens when their use is therapeutic.

On the other hand, intravenous immunoglobulins for the treatment of immunodeficiencies and hyperimmune immunoglobulins for passive immunization therapies are obtained from blood plasma from human donors and their supply is limited. There is no recombinant alternative at present.

The problems of obtaining / producing / using polyclonal antibodies, antivenoms based on antisera, immunological sera for passive immunity obtained from immunized animals, or intravenous immunoglobulins for the treatment of immunodeficiencies and hyperimmune immunoglobulins for passive immunization therapies that obtained from blood plasma from human donors, they could be solved by recombinant production of complex repertoires of polyclonal antibodies. However, there are numerous technical problems that limit this possibility. Some strategies in this regard have been developed based on transgenic animals. Such is the case of transgenic mice that have been introduced with human immunoglobulin loci (US 6,11,116), which can produce human polyclonal antibodies by conventional immunization techniques. However, this system produces minimal amounts of antibodies due to the small size of the animals. Transgenic animals of larger size have also been obtained as cows (Kuroiwa et al., 2002).

However, transgenic animals have certain drawbacks. First, the instability of immunoglobulin loci compromises the production of antibodies in the long term. Secondly, it is difficult to eliminate the immunoglobulins of the animal itself. Third, transgenic animals are not exempt from the risk of inadvertent transmission of pathogens. Finally, the composition of the mixture is not entirely reproducible because it will depend on the immunophysiological conditions of the animal.

An alternative to transgenic animals is to produce recombinant polyclonal antibodies from variable sequences of immunoglobulins obtained from individuals with an established immune response, using mammalian cells as a production platform. This technology uses adapted mammalian cell lines to achieve a site-specific integration of each of the transgenes that constitute the sample (Bregenholt et al., 2006; Haurum, 2006; Klitgaard et al., 2006; Koefoed et al., 2011 ; Koefoed et al., 2006; Pedersen et al., 2010; Wiberg et al., 2006). This eliminates the variability of expression due to the positional effect of the transgene and facilitates the production of reproducible mixtures of polyclonal antibodies with high batch-to-lot consistency on an industrial scale (WO 2004/061 104).

More recently, a protocol has been developed that by individual selection of stable clones of mammalian cells transformed by random insertions allows polyclonal mixtures to be produced with batch-to-batch reproducibility (WO 2008/145133). However, these methodologies require a laborious process of selecting stable individual clones, so it is poorly suited for highly complex polyclonal mixtures. In addition, systems based on mammalian cells have high costs of investment, production, and industrial scaling, which makes the use of alternative platforms that reduce costs attractive. A particularly interesting group of alternative platforms are those based on plants. The plants are used as biofactories of proteins of very different nature using, among other methodologies, recombinant viruses (Orzaez et al., 2009). There are numerous examples of plant viruses that have been adapted to the production of recombinant proteins in plants (Gleba et al., 2004). For this, the wild virus undergoes a series of modifications aimed at converting it into a viral vector, that is, a nucleic acid, generally of a plasmidic nature capable of transferring self-replicating units to the cell that house the sequence encoding the protein of interest. One of the main advantages of the use of viral vectors is that their self-replication capacity allows amplifying the number of copies of the transgene in the cell, which favors obtaining high levels of recombinant production. There are currently numerous viral vectors being exploited by academic laboratories and biotechnology companies (for example WO 05/049839, WO 06/079546).

So far, viral vectors have been used for the production of homogeneous products, such as monoclonal antibodies, some of which are in clinical trial phases. A precedent in the attempt to move towards the production of polyclonal mixtures of antibodies based on viral systems of plants appears in a work developed by Julve et al., 201 1 (Master thesis summary, Polytechnic University of Valencia). Specifically, reference is made to the production of a polyclonal mixture of Nanobodies (VHHs) using Nicotiana benthamiana leaves. However, the methodology summarized in Julve et al., 2011 is very laborious as it is based on the individualized expression of each antibody in separate and independent leaves and their subsequent mixing and purification, resulting in a final polyclonal mixture. Therefore, it is a strategy based on mono-transgenic plants that are subsequently mixed. The Julve et al., 2011 approach does not provide any procedure to produce recombinant protein mixtures of high or very high complexity, since these are inaccessible to a method that does not involve simultaneous expression.

Taken from this unique precedent, to date no viral vectors have been used for the simultaneous production of complex mixtures of recombinant proteins, among other reasons because it is considered that, according to the commonly accepted quasi-species model, the competition that takes place between the different Clones that co-infect a plant leads to the predominance of a few clones over others. Is expected therefore, the resulting mixture has a unique and non-reproducible composition, since the concept of "competition between clones" leads to the dominance of some variants over others and therefore to a very low reproducibility between experiments. The reproducibility requirement would be very advantageous, since it would allow the production of therapeutic mixtures with constant batch-to-batch composition.

Therefore, there is currently a need to develop a method for the production of complex repertoires of recombinant molecules, based on plants and tissues that meet the requirements of "multi-transgenesis" (co-expression of the different transgenes simultaneously in the form of a somatic mosaic in the same tissue) and reproducibility of the expression system (production of highly reproducible mixtures between independent experiments).

BRIEF DESCRIPTION OF THE INVENTION

The present invention describes a method for obtaining polyclonal mixtures of recombinant molecules, in a consistent and reproducible manner, by transiently generating multi-transgenic plants that give rise to somatic mosaics induced by viral replicons that exclude each other.

Current eukaryotic platforms do not offer the possibility of producing complex repertoires of proteins, since they are mostly based on the introduction into the recombinant organism of a single gene (or at most a small group of genes in the case of oligomeric proteins). The production of polyclonal mixtures presents technical difficulties, which are different depending on the type of platform used.

(i) In the case of complete organisms, such as animals or transgenic plants, the technical problem is the difficulty of transforming the genome stably with a high number of similar sequences. To this we must add the difficulty of controlling the expression levels of each individual gene integrated in the genome, which makes it difficult to obtain a defined mixture.

(ii) In the case of cell cultures, competition between clones during the growth phase of the crop leads to loss of diversity and therefore poor reproducibility. An alternative to obtain composition mixtures defined consists of producing separately the different components individually and mixing afterwards! ^' . This implies maintaining parallel production and purification lines, which implies an increasing cost that is unfeasible when the complexity of the mixture reaches a certain threshold.

The present invention is based on a little studied aspect of viral infection in plants: the high spatial structuring of the clonal variants produced during the course of the infection (Elena et al., 201 1; Sardanyes and Elena, 201 1). This spatial distribution occurs as a consequence of the anatomical structure of the plants and is facilitated by a phenomenon known as "exclusion of superinfection" or "homologous interference." This phenomenon is that the presence of a "resident" viral clone in a cell or group of cells prevents their superinfection by a second "challenger" viral clone highly related to the first (Folimonova, 2012; Syller, 2012; Ziebell and Carr, 2010). This phenomenon is especially evident when experimentally infecting a plant with a complex mixture of viral variants. In this case, the different variants do not enter into competition among all of them as one would expect, which would lead to a homogeneous distribution led by the most suitable clone. On the contrary, each variant colonizes a region of the plant excluding the others and giving rise to a spatial distribution structured in mosaic that allows the coexistence in different cells of the same plant of the different clonal variants.

This invention describes the use of the natural phenomenon of "superinfection exclusion" or "homologous viral interference" for the production of spatially delimited sets of proteins (somatic mosaics) that can give rise to polyclonal mixtures of recombinant products of defined, reproducible composition and With high yields.

The invention makes use of viral vectors derived from a plant virus. Until now, the main advantage conferred by viral vectors is that they allow high yields of recombinant protein to be achieved. This is due to its capacity for self-replication, which in turn leads to the amplification of the gene that encodes the recombinant protein to be produced. There are many viral vectors described in the field literature that adapt to this description and are therefore usable to develop the method of the invention. Said viral vector may be formed either by a complete viral genome or by a viral genome from which superfluous parts have been removed, but that retains the basic functions of the wild virus, and at least the self-replication function, the function of cell-to-cell movement and the ability to exert "homologous interference" between related clones. Said viral vector must also be able to recombinantly incorporate a nucleotide sequence that encodes the gene (s) of interest and induce its translation into protein in the host cell. With the present invention that homologous interference property becomes as important as the high levels of production.

In the present invention, the production of a mixture formed by an N number of different recombinant proteins is performed by first cloning the N genes that encode the N proteins of interest at the same position within the viral vector. Cloning can be performed clone by clone separately, or performed together in a single cloning reaction in which all the gene variants of interest are included in the same solution. Once the N clonal variants of the viral vector have been generated, they are transferred jointly and simultaneously to the host plant using one of the different methods of transferring viral vectors accessible in the state of the art, in a dilution such that entry is favored. of an average number between zero and one of replicative entities per host cell.

In a preferred but not limiting embodiment of the present invention, the transfer of the N clonal variants of the viral vector to the plant cells is carried out by means of transient genetic transformation mediated by Agrobacterium tumefaciens of an infectious clone of the virus, thus generating a transiently plant multi-transgenic Under these circumstances, one or none of the clonal variants of the viral vector is mostly replicated in each host cell. As a consequence of the cell-to-cell movement function, and possibly the systemic movement function of the viral replicon, each viral clone travels from the initially infected cell to adjacent and / or distant cells that have not in turn been primarily infected by another viral clone, amplifying its genome there and accumulating the product of the recombinant gene. However, by virtue of the homologous interference function, each viral clone is excluded from those cells in which there is an infection already established by another resident clone. As a result, a structured mosaic distribution of the different viral clones is obtained where the relative abundance of each clone is a function, among other parameters, of its ability to move from cell to cell. Homologous interference causes a structured mosaic distribution of the Clonal variability that minimizes the effect of competition between clones. After the time necessary for a stable mosaic to develop, the plant is homogenized and the extraction and / or purification processes are carried out, depending on the nature of the proteins to be produced, resulting in a complex mixture. of recombinant proteins whose composition is consistent and reproducible between experiments.

The population of recombinant proteins produced changes during the early stages of infection, but given an incubation time long enough for all plant cells to have colonized, the relative abundance of each protein in the mixture (Ci) is a function of the following parameters: (Vi) the cell-to-cell movement speed of each clone; (Pi) the specific protein production levels of each clone; (ODRi) the relative abundance of each clone in the initial inoculum.

In principle, the co-existence of two or more clones in the same cell makes it necessary to include a fourth factor (lij) that reflects in its case the result of the interaction between two clones i and j when they compete for the biosynthetic resources of the same cell , lij is a complex, different parameter for each pair ij. The complexity of the lij parameter makes Ci highly variable and unpredictable in systems without homologous interference. However, in the methodology described in this invention, the existence of a homologous interference phenomenon minimizes the interaction between clones, allowing the lij parameter to be neglected and therefore facilitating the predictability and reproducibility of the expression of polyclonal mixtures.

Therefore the described system allows the production of mixtures of high or very high complexity and even of mixtures of indefinite composition with high yields. The theoretical limit of different components that said mixture can comprise is of the same order as the total number of cells used in the production process. Since the methodology is scalable simply by increasing the number of plants, the potential complexity of the mixture is virtually unlimited. Alternatively, this methodology can be used for the production of a defined polyclonal mixture comprising N clones previously selected.

The reproducibility requirement is very advantageous, since it would allow to produce therapeutic mixtures with constant composition batch to batch. The process of the invention gives place to mixtures whose composition is highly reproducible between experiments. The high reproducibility of the multitransgenic expression of the process of the invention is demonstrated in the examples, where experiments are described in which mixtures of very high complexity (> 100 elements) are tested. It is pointed out that the possible reason for this reproducibility is the implementation of a homologous interference phenomenon, although this criterion is different from that described in the state of the art (the connection between homologous interference and reproducibility phenomena has not been established at the moment. ) and therefore constitutes a valid alternative to the previous methods of producing complex mixtures of recombinant proteins.

In addition to the aforementioned reproducibility, the method of the invention has an important application in the production of polyclonal multiprheoretic complexes, since homologous interference prevents the formation of multiprheretic complexes other than those desired. Thus, when the clones to be expressed form homopolymers, such as homodimers or homotetramers, the co-expression of two or more clones in the same cell by traditional methods leads to the formation of an inconsistent mixture of heteropolymers. In contrast, the process of the invention favors the preferential formation of homodimers. Likewise, the method of the invention facilitates polyclonal expression of mixtures of heteropolymers of defined composition, as is the case of polyclonal antibodies.

In this particular case, each heavy chain has an accompanying light chain. When expressing polyclonal mixtures in systems without homologous interference, a phenomenon known as "chain shuffling" takes place, whereby each heavy chain can be joined with any light chain other than its companion, giving rise to an antibody of suboptimal activity. The method of the invention minimizes the effect of shuffling chains by minimizing interference between clones.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a method for the production of at least one complex repertoire of N recombinant molecules, characterized by comprising the following steps: i) assembling a collection of N distinct nucleotide sequences in a viral vector of destination derived from a plant virus that in its infection mechanism presents the property known as "homologous interference" or "exclusion of superinfection", in such a way that a collection of viral clones is generated; ii) transfer of the collection of viral clones jointly and simultaneously to a host plant or an organ or tissue of a host plant that is capable of harboring replication and movement of the viral vector. iii) generation of a transiently multi-transgenic plant structured in the form of a somatic mosaic where more than 80%, preferably more than 90%, of the cells express exclusively one of the N recombinant molecules, or express it very largely, in a proportion molar representing at least 80%, preferably at least 90%, of the total of N recombinant molecules produced in that same cell.

In a preferred embodiment of the present invention, the method described above is characterized in that the N recombinant molecules are N recombinant proteins. In this case, step i) is characterized by assembling a collection of N distinct nucleotide sequences that correspond to the coding sequences of each of the different N proteins that are to be produced in polyclonal format, in a viral destination vector derived from a plant virus that in its mechanism of infection has the property known as "homologous interference" or "exclusion of superinfection", in a way that allows its translation into proteins, forming a collection of viral clones. Likewise, step ii) is characterized by the transfer of the collection of viral clones jointly and simultaneously to a host plant or an organ or tissue of a host plant that is capable of harboring replication and movement of the viral vector, as well as the translation of the N recombinant proteins.

In another preferred embodiment of the present invention, the method described above is characterized in that the viral vector of destination of step ii) is in the form of an infective clone inserted in a binary vector and uses the transient transformation based on Agrobacterium tumefaciens as a mechanism of entry into the cells of the host plant thus generating a multi-transgenic plant that expresses the N recombinant molecules, preferably N recombinant proteins, in the form of a somatic mosaic. Preferably, the viral vector is devoid of its movement function systemic More preferably, the viral vector is delivered to the host plant cells by a mechanical system. Even more preferably, the mechanical system is selected from the following group: vacuum infiltration, overpressure infiltration and spray.

In another preferred embodiment of the present invention, the method described above is characterized in that the N nucleotide sequences of step i) encode at least one sequence variant of a family of proteins. Preferably, the family of proteins is selected from the following group: enzymes, immunoglobulins, membrane receptors, intracellular receptors, and lectins.

In another preferred embodiment of the present invention, the method described above is characterized in that it is combined with an expression system without homologous interference that allows the production of heteromultimeric proteins where some of the components of the multimer are variable and are produced by the system with interference, while other components are constant and are produced by the non-interfering system.

In another preferred embodiment of the present invention, the method described above is characterized in that the N nucleotide sequences of step i) encode at least one variable sequence of at least one immunoglobulin. Preferably, the immunoglobulin is produced by lymphocytes obtained from animals immunized against an antigen.

In another preferred embodiment of the present invention, the method described above is characterized in that the N nucleotide sequences of step i) encode at least one variable sequence of at least one immunoglobulin or its fragment, selected by an in vitro antibody selection technique recombinants (phage display).

The present invention also refers to the use of the method described above, to recombinantly produce a complex mixture of homopolymers.

Likewise, the present invention also refers to the use of the method described above to recombinantly produce a complex mixture of heteropolymers where each cell produces a single type of heteropolymer, minimizing the effect of "chain shuffling".

The present invention also refers to a transiently multi-transgenic plant or a fragment thereof, characterized in that it is obtained in step iii) of the method described above, plant or plant fragment (organ, tissue) transfected with a collection of viral clones as described in step ii) of the method described in claim 1, and expressing a polyclonal mixture of proteins.

Preferably, the plant or fragment thereof expresses in whole or in part and recombinantly, at least one complex repertoire of polyclonal antibodies. More preferably, polyclonal antibodies are similar to those produced by an animal or a group of animals in response to an immunization process against an antigen, a group of antigens or a pathogen.

The present invention also refers to a crude extract obtained from a plant described above.

Likewise, the present invention also refers to a purified or partially purified mixture of antibodies obtained from a crude extract described above.

Finally, the present invention also refers to a complex repertoire of N recombinant molecules, characterized in that it is produced by the method described above. Preferably, the N recombinant molecules are N recombinant proteins. More preferably, the N recombinant proteins are selected from the following group: polyclonal antibodies, antiserums based on antisera, immunological sera for passive immunity, and intravenous immunoglobulins.

The method of the invention begins with the cloning of DNA fragments encoding recombinant proteins in a viral vector derived from a plant virus such as TMV, PVX, CTV, CMV or any other virus that exhibits homologous interference property.

In a particular embodiment of the technique, the vector is derived from a single stranded RNA virus. In another particular embodiment the viral vector consists of an RNA virus of simple chain that maintains in its genome the functions of replication and cell-to-cell movement but has eliminated the systemic movement function. The systemic movement, which takes place through the vascular bundles, can generate bottlenecks in the extension of the viral clones throughout the plant, so that their suppression facilitates the reproducibility of the technique.

The transfer of the DNA fragments to the viral vector can be performed by any of the techniques for assembling DNA fragments accessible to the state of the art, such as digestion / ligation with type II restriction enzymes, cyclic digestion / ligation with type restriction enzymes US, site-specific recombination, independent ligase cloning, homologous recombination or PCR assembly techniques.

The DNA fragments can be incorporated, either one at a time, or in a group, generating a library of viral vectors, each comprising a different sequence.

In a particular embodiment of the technique, the different DNA fragments can encode different complete antibodies comprising different variable sequences from humans, mice, camelids or other mammals.

In another particular embodiment, the DNA fragments may encode scFv or VHH antibody fragments from humans, mice, camelids or other mammals.

In another particular embodiment, each viral vector can incorporate two or more DNA fragments in two different positions of its genome, encoding, for example, a heavy chain of an antibody and the corresponding light chain of the same antibody so that the co-expression of both in the same cell give rise to a functional combination.

In all cases, the final result will be a collection of F clones of the viral vector containing fragments or combinations of recombinant DNA fragments that together represent N distinct sequences, where N is any value between 2 and F.

Next, the F viral clones comprising N distinct recombinant sequences they are transferred jointly and simultaneously to the plant cells of the host plant using some of the mechanisms of the state of the art to induce the formation of viral replicons. The host plant can be any plant species capable of harboring a viral replicon. It can be a complete plant or an explant of it as a leaf or other organ such as a fruit or a root. In a particular embodiment, the host plant is a plant of the Nicotiana genus. In another particular embodiment the host plant belongs to the Nicotiana benthamiana species.

In a particular embodiment, the joint transfer of the various viral clones to the cells of the host plant may be mediated by the Agrobactenum bacteria. Said bacterium is capable of transferring a DNA fragment (T-DNA) that comprises the gene or genes of interest to the plant cell, so that they are produced by the plant cell. The transfer is transient because it does not require integration of the T-DNA into the chromosome of the cell, so the T-DNA is not transferred to the germ line and its expression lasts a few days. In this particular embodiment, viral replicons can be introduced entirely into the T-DNA to be transferred in the form of a copy DNA introduced into the bacteria's T-DNA. In this way, Agrobactenum functions as a shuttle to transfer the viral replicon into the cell.

To obtain the polyclonal production of recombinant proteins using Agrobactenum tumefaciens as a shuttle, the viral vector library must preferably be integrated into a binary vector containing a T-DNA, so that the viral sequence is under the regulation of a promoter that It operates in the host cell. Each of the clones of the binary vector collection is transferred, either one at a time, or together, to Agrobactenum tumefaciens cells, generating a polyclonal collection of Agrobactenum strains. This process can be done directly, either through an intermediate step in Escherichia coli that facilitates the transformation process. In this second case, the library is first transferred to Escherichia coli cells, resulting in a collection of independent colonies. Subsequently, colonies are grown independently or together under conditions that minimize the loss of diversity, isolating from these colonies the collection of binary plasmids. From this last preparation, the competent cells of Agrobactenum tumefaciens are transformed into a "seed" collection of Agrobactenum. The "seed" collection comprises one or several Agrobacterium cultures obtained from the initial transformation of the binary plasmid collection into a preparation of competent Agrobacterium cells. The collection can be maintained in the form of F Agrobacterium cultures, each housing one of the F clones, kept separately and stored in freezing conditions. In this case F must be greater than or equal to N. This option is the usual one for a small and known N number, such as 5, 10, 20 or 100 different sequences, in which case F is usually equal to N. It is also the usual case for a small but unknown N number, such as 5, 10, 20 or 100 different sequences, in which case F is usually greater than N. Alternatively, when N is a large number, generally greater than 10 and usually greater than 50 e even greater than 1000, the "seed" collection can be conserved in the form of a joint stock maintained under freezing or freeze-drying conditions containing a number F of viable bacteria, where F is much greater, at least an order of magnitude greater than N.

To obtain a recombinant polyclonal mixture, the Agrobacterium seed culture is grown long enough to at least double and usually multiply by 5 or even 10 or more the initial number of cells contained in the initial aliquot. This culture is then transferred to a suitable buffer solution, it is conveniently diluted to obtain the infiltration mixture, which is subsequently inoculated at some discrete point of the host plant by means of some of the agroinoculation technologies available in the state of the art.

In a particular embodiment, viral clones are devoid of systemic movement capacity. In this case, the infiltration mixture is artificially delivered to the largest possible number of host cells. For this, the Agrobacterium cells present in the infiltration mixture are contacted with the host plant cells so that the former are competent to transfer their T-DNA to the latter efficiently. This can be done through agroinfiltration, by vacuum immersion or by any other system available to the state of the art, such as spray techniques, use of abrasives, surfactants, etc.

Once the T-DNA reaches the nucleus of some cells, the replicon is transcribed, giving rise to an active viral genome that replicates in the initial cell and is transferred to neighboring cells. Then the plant infected by the polyclonal mixture of replicons it is maintained in favorable conditions of light and humidity for a variable period of time that includes between a minimum of 4 days and the complete plant growth cycle; During this time the viral clones replicate, move cell to cell until they find a cell occupied by another replicon, and translate the protein of interest whose information they house in their genome. Together, the end result is a mosaic distribution of the different clones across the plant surface. In said mosaic, each tile is formed by a variable number of cells. The inner cells of the tile express exclusively one of the recombinant proteins of the mixture, or at least express it in a very large molar proportion, where the range of contamination with other proteins in the mixture is less than the limit of detection of fluorescent proteins in a confocal microscope and in any case less than 10% in molar ratio. Exceptionally, some cells located on the border between two adjacent tiles can express more than one recombinant protein in molar proportions close to 50%. The total number of these border cells capable of simultaneously expressing more than one recombinant protein is a function of the average size of the tiles. The average size of the tiles depends on the concentration of Agrobacterium cells in the infiltration mixture, and by extension of the total optical density of the culture (ODT), this parameter being experimentally modulable. Together, the maximum number of cells susceptible to co-expressing detectably more than one fluorescent protein can be experimentally adjusted to be less than 10%, or less than 5% and possibly less than 1%.

After the incubation time, the plant material is harvested and the set of proteins of interest is purified using a purification method that is common to all components of the mixture, obtaining a polyclonal mixture of proteins of defined composition.

The present invention allows the size and number of tiles of the expression mosaic to be modulated by manipulating the optical density of the infiltration mixture. Thus, infiltration mixtures of N clones with ODT of 0.1 result in larger tiles than infiltration mixtures containing the same N clones at an ODT of 0.033. In turn, infiltration mixtures of N clones with ODT of 0.033 give rise to larger tiles than infiltration mixtures containing the same N clones at an ODT of 0.01. Large tiles containing on average several tens or even Hundreds of cells need lower concentrations of Agrobactenum, which facilitates the elimination of possible toxins associated with this bacterium. In addition, the use of large tiles minimizes the residual interaction between clones, since this takes place primarily in the border zone between adjacent tiles. On the other hand, the use of small tiles, comprising a few cells or tens of cells on average, facilitates the expression of a greater number of clones per plant, favoring polyclonal diversity. Likewise, the use of small tiles formed by a few cells or dozens of cells minimizes the influence of the speed of movement (Vi) on the final composition of the mixture.

In the present invention reference is made to a "seed" collection, which comprises a number N of distinct and known clones, conserved separately in the form of N cryopreserved cultures. Likewise, the present invention allows modulating the final composition of the polyclonal mixture by manipulating the composition of the infiltration mixture. Given a sufficiently long incubation time for all plant cells to have colonized, the relative abundance of each protein in the mixture (Ci) will be a function of the following parameters: (Vi) the cell-to-cell movement speed of each clone; (Pi) the specific protein production levels of each clone; (ODRi) the relative abundance of each clone in the initial inoculum. Being Pi and Vi constant and specific values for each clone, it is possible to manipulate the composition of the mixture by modifying the ODRi values for each clone. For this, the N seed clones are grown separately long enough to at least double and usually multiply by 5 or even 10 or more the initial number of cells contained in the initial aliquot. Next, the optical density of each crop is determined and the different cultures are mixed in defined proportions to give rise to the final infiltration mixture. The manipulation of the relative ODs of each clone allows the final composition of the mixture to be modulated significantly and reproducibly, since this parameter determines the relative abundance of the initial foci that infect the plant for each clone.

In a particular embodiment of the technique, polyclonal expression based on viral vectors with homologous interference is combined with another expression system that does not exhibit the phenomenon of homologous interference. In a particular embodiment, the second "non-interfering" system consists in the Agrobactenum-mediated transfer of a T-DNA that does not comprise any autorepliative structure. This Particular embodiment allows to produce recombinant multiprheoretic complexes in which one or more of the components of the mixture are variable and are expressed through the use of a platform with homologous interference, and the rest of the components are constant for all clones and are connected. express through a system without homologous interference. In another particular embodiment of the mixed process, the variable part of the multiproteic complex is constituted by a polyclonal mixture of antibodies or antibody fragments, while the constant part is formed by a constant peptide that mediates the formation of polymeric antibody structures such as the chain. J, or by a structure that protects antibodies against proteolytic degradation as the Secretory Component.

In the present invention, the term "complex repertoire of N recombinant molecules" refers to N recombinant DNA or RNA molecules, N recombinant proteins encoded by said DNA or RNA molecules, or the metabolites resulting from the activity of said N recombinant proteins . Preferably, this term refers to a set of more than 10 DNA or RNA molecules that differ from each other in at least one nucleotide of their nucleotide sequence or a set of more than 10 different proteins that differ from each other in at least one amino- acid from its peptide sequence and that are produced in an organism other than those that are original by recombinant DNA strategies.

In the present invention, the term "N recombinant proteins" refers to N enzymes, N immunoglobulins, N membrane receptors, N intracellular receptors, N lectins N polyclonal antibodies, N antivenoms based on antisera, N immunological sera for passive immunity, and No intravenous immunoglobulins.

In the present invention, the term "collection of N distinct nucleotide sequences" refers to a set consisting of an indeterminate number of DNA or RNA fragments that differ from each other at least in a nucleotide position within their sequence.

In the present invention, the term "target viral vector" refers to a nucleic acid derived from a virus that encodes the functions necessary for reproduction in the host cell and into which those nucleic acid fragments that are recombinantly integrated It is intended to express recombinantly. In the present invention, the term "viral clone collection" refers to a set of nucleic acids derived from a virus that differ in sequence in at least one nucleotide.

In the present invention, the term "host plant" refers to any plant organism in whose cells replication of a virus or a viral vector can take place.

In the present invention, the term "transiently multi-transgenic plant structured in the form of a somatic mosaic or a fragment thereof" refers to a complete plant or a fragment of a plant comprising a set of more than two cells, in the which has been introduced through genetic transformation techniques a set of transgenes in such a way that each individual cell receives and expresses zero, one or more of a transgene, which transgenes are not necessarily identical to those received and expressed by the rest of the cells of the set, and in which the package of zero one or more received genes may or may not be stably integrated into the genome and therefore may or may not be transmitted to the progeny and cease to express themselves in said cells after a certain time.

In the present invention, the term "infectious clone inserted into a binary vector" refers to a DNA sequence derived from a plant virus that is inserted into a binary vector and which, when transferred to the nucleus of a cell, is capable of inducing its own transcription giving rise to an RNA molecule capable of infecting the host cell.

In the present invention, the term "transient transformation based on Agrobacterium tumefaciens" refers to a transfer of one or more DNA molecules from the cytoplasm of the bacterium Agro bate rium to the nucleus of the plant cell, which occurs transiently without that the stable integration of said DNA molecules or part thereof into the chromosomal DNA of the host cell necessarily occurs.

In the present invention, the term "systemic movement function" refers to the ability of a plant virus to move long distances within the host plant traveling through its vascular system.

In the present invention, the term "mechanical system" refers to any a procedure that involves the application of a force, an external pressure or an electric current or any other membrane permeabilization mechanism to favor the entry of a DNA into the host cell.

In the present invention, the term "sequence variant of a family of proteins" refers to each of the individual variants that can be presented in a set of proteins of the same family.

In the present invention, the term "expression system without homologous interference" refers to any recombinant protein production system that is based on a vector that naturally does not exhibit the phenomenon of homologous interference, that is, in the case If two or more clones of said vector colonic the same cell, they do not necessarily exclude each other so that none of them necessarily prevails over the others.

In the present invention, the term "variable immunoglobulin sequences" refers to regions of immunoglobulin proteins that exhibit high sequence variability and where their antigen binding capacity resides.

In the present invention, the term "variable sequences of immunoglobulins or their fragments selected by an in vitro selection technique of recombinant antibodies such as phage display" refers to all or part of one or several variable regions of immunoglobulins selected for their binding ability to a particular antigen or set of antigens by in vitro selection procedures.

In the present invention, the term "complex repertoire of polyclonal antibodies" refers to a set consisting of a greater number of ten different immunoglobulin proteins, where the difference between them lies, at least partially, in the amino acid sequence of their variable regions.

In the present invention, the term "crude extract" refers to a complex mixture of compounds, obtained from a plant or plant tissue and obtained by an extraction process that includes the breakdown of plant tissue cells from from which it is obtained and the solubilization of the cellular compounds released in a solvent suitable.

In the present invention, the term "purified or partially purified mixture of antibodies obtained from a crude extract" refers to a set of N distinct antibodies, which differ from each other at least in one amino acid of their peptide sequence, isolated from of a crude extract of the plant material where they are produced recombinantly under conditions that allow the total or partial elimination of the rest of the cellular compounds present in the extract.

In the present invention, the term "complex repertoire of antivenoms based on antisera" refers to a set of immunoglobulins obtained from sera from animals immunized with a toxic substance.

In the present invention, the term "complex repertoire of immunological sera for passive immunity" refers to a set of immunoglobulins obtained from sera of animals immunized against a pathogen or a toxic substance and which are effective in protecting a receiving organism against said pathogen or said substance, without the need to activate an immune response in the receiving organism.

In the present invention, the term "complex repertoire of intravenous immunoglobulins" refers to a set consisting of an indeterminate number of distinct immunoglobulins that can be delivered to an animal or a human being intravenously to produce some kind of benefit.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES Figure 1. Individualized production of VHHs. (A) Alignment of amino acid sequences of the 9 VHHs produced in N. benthamiana plants: SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 8, SEQ. ID. No. and SEQ. ID. No. 9. (B) Western Blot analysis of the different VHHs produced individually.

Figure 2. Production of an oligoclonal repertoire of VHHs in a transiently oligo-transgenic plant. (A) SDS-PAGE analysis of purified fractions (E1 and E2) and final washing (F) of a repertoire of 9 VHHs produced in a transiently oligo-transgenic plant of the invention. Discreet protein banding is revealed with Coomassie blue. (B) 2D electrophoresis with development of silver nitrate of fraction E2.

Figure 3. Production of a polyclonal repertoire of VHHs in a transiently multi-transgenic plant. (A) SDS-PAGE analysis of purified fractions (E1 and E2) and the last wash (F) of a repertoire formed by an indefinite number of VHHs (F> 10 ⁴ ) produced in a transiently multi-transgenic plant of the invention. Continuous protein banding is revealed with Coomassie blue. (B) 2D electrophoresis with development by means of differential fluorescent marking (DIGE) of three E2 fractions corresponding to three independent polyclonal VHHs production experiments. The three experiments were carried out from the same seed crop of Agrobacterium tumefaciens that houses a library of VHHs obtained from sera of non-hyper-immunized camels.

Figure 4. Scatter plots of purified polyclonal VHH fractions produced in three independent experiments. The axis of bees represents the intensity ratio between two experiments for each spot. The ordinate axis represents the absolute intensity (volume) of each spot in the DIGE experiment.

Figure 5. Relative frequency graphs of the 100 most abundant sequence variants obtained by massive Ion Torrent sequencing. The percentages represent the relative frequency of each individual unigen versus the total unigenes of their sample. (A) Frequency distribution shows EXP1 (B) Frequency distribution shows EXP2.

Figure 6. In vitro anti-venom activity measured by ELISA assays of the different VHH repertoires obtained from multitransgenic plants transformed with libraries from preimmune (C1 P, C2P and C3P) and hyperimmune (C1 H, C2H and C3H) camel samples. (A) Activity of the different samples tested against Crotalus simus venom and against BSA (B) Comparison of the activity of hyperimmune samples against Crotalus escutulatus venom and against unrelated cobra venoms (N. ninubiae and N mossambica). Abscissa axis represents the absorbance at 492 nm, and in the axis of ordinates (A) dilutions of VHH extracts, (B) serial dilutions VHH (micrograms).

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the efficacy of the invention in producing multitransgenic plants as well as the reproducibility of the method for the production of complex repertoires of recombinant molecules, preferably recombinant proteins, object of the invention. present invention Specifically, the tests carried out show the production of complex repertoires of N recombinant proteins. The existence of N recombinant proteins necessarily implies the presence of N recombinant DNA and RNA molecules precursors thereof. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1. Production of an oligoclonal mixture of camel nano-antibodies (VHHs) in transiently oligo-transgenic N. benthamiana plants

In order to demonstrate the possibilities of polyclonal expression based on homologous interference for the production of mixtures of recombinant molecules, preferably recombinant proteins of industrial or pharmaceutical interest, a small collection of clones of simple camelid antibody fragments was preferably used and not limited chain (also called nanobodies or VHHs).

In this example, the possibility of simultaneously producing a discrete number (N <10) of recombinant proteins was tested giving rise to a mixture of composition defined (oligoclonal) where the identity of each and every one of the proteins in the mixture is known. For this, a population of VHHs was amplified from a cDNA preparation obtained from a fraction of nucleated camel blood cells using oligonucleotides VHHF (SEQ. ID. No. 10) and VHHR (SEQ. ID. No. 1 1 ). From said amplification, the VHHs were transferred to the pGTMV vector (SEQ. ID. No. 12) using a single fragment GoldenGate reaction (Engler et al., 2009; Engler et al., 2008; Sarrion-Perdigones et al., 201 1) mediated by T4 ligase and the restriction enzyme Bsal in a 35 cycle incubation. In this way the nine different clones (pGTMV_VHH 1 to pGTMV_VHH9) that were used in the oligoclonal expression were isolated. All constructs contained a fusion to a tail of six histidines incorporated by the pGTMV vector. Nucleotide sequences of the coding regions of each of the 9 cloned VHHs are shown as SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. I D. No. 7, SEQ. ID. No. 8, SEQ. ID. No. and SEQ. ID. No. 9. An amino acid alignment of the VHH sequences involved in the experiment is shown in FIGURE 1A.

First, the individualized expression of the different VHH was tested. All clones derived from pGTMV were transferred to Agrobacterium tumefaciens and preserved as seed crops. Subsequently, an aliquot of 10 μΙ_ of each of the seed cultures was thawed and grown at 28 ° C under stirring (200 rpm) for 18h in 1 mL of LB with 12μg / mL of Carbencillin and 50 µg / mL of Rifampicin. Then, 100 μ \ - of each culture was transferred to 5 mL of LB with 12μg / mL of Carbencillin and 50 µg / mL of Rifampicin and grown for 18h at 28 ° C under stirring (200 rpm). The resulting cultures were centrifuged at 500 g for 15 minutes and the cell precipitate was resuspended in 10 mL of infiltration buffer (10mM MES pH 5.6, 10mM calcium chloride, 2μΜ acetosyringone).

Similarly, we proceeded with Agrobacterium cultures that house the 5 ^' deconstructed TMV module (plCH 17388) and the integrase module (PICH 14011) necessary for the establishment of the viral replicon (Marillonnet et al., 2004a). The optical density (OD) of each Agrobacterium culture was measured and the relevant dilutions were made in infiltration buffer to bring each culture to an OD = 0.1 measured in a spectrometer. Nine mixtures were subsequently made, one for each VHH (pGTMV_VHHi: plCH17388: PICH14011 in a 1: 1: 1 ratio), and each mixture was infiltrated separately into the intercellular spaces of Nicotiana benthamiana leaves by means of a 2 ml syringe without needle. 3 leaves per sample were infiltrated, using different plants. During the days following the infiltration, the appearance of a necrosis phenomenon similar to the hypersensitive response (HR) was observed in some clones, so it was decided to advance the collection process prior to the appearance of symptoms (6 dpi) and collect all the plants in that phase. After collection the samples (whole leaves) were weighed and frozen in liquid nitrogen and stored in a freezer at - 80 ° C. To extract the plant material, the plant material was crushed in a mortar in the presence of liquid nitrogen with PBS buffer in a 1: 3 ratio (tissue weight: buffer volume). Subsequently, the crude extract was centrifuged at 10 krpm for 15 min at 4 ° C and the supernatant was filtered twice with filter paper and once more in steriCup filters. The resulting extract was passed through a Ni Agarose affinity column and the purified proteins were eluted in 50 mM phosphate buffer at pH4.0 and the elution fraction was immediately neutralized with 1/5 volume of PBS. A 6 μΙ aliquot of each elution was resolved in a PAGE electrophoresis and revealed by Western blot using a murine primary anti-histidine antibody followed by a secondary anti-mouse antibody conjugated to horseradish peroxidase. The result of said Western blot can be seen in FIGURE 1 B. All the VHHs tested were expressed and purified in N. benthamiana, although with different yields. Thus, VHH4, VHH5, VHH6, VHH7, VHH8 and VHH9 were produced at levels between 80 and 120 μg of purified protein per gram of fresh weight, while for the remaining three VHHs levels of between 2 and 10 μg of protein were obtained. per gram of fresh weight.

The oligoclonal expression of the nine VHHs was then tested. For this, the seed cultures were grown individually as described in the previous experiment, but in this case all the cultures were combined in a single mixture of infiltration formed by the cultures (pGTMV VHHI: pGTMV_VHH2: pGTMV_VHH3: ...: pGTMV_VHH9 .plCH17388: PICH14011) in proportion (1: 1: 1: ...: 1: 9: 9). A single infiltration was made in three leaves of three different N. benthamiana plants. After 6 dpi, the plant material was collected and the recombinant proteins were purified following the procedure described above. As a result, a mixture of VHHs was obtained with a yield of 80 μg of purified protein per gram of fresh weight. The mixture was resolved by PAGE electrophoresis as shown in FIGURE 2A. This figure shows the runs corresponding to the last wash (F) and two successive elutions (E1 and E2) of the purification process. In said electrophoresis, revealed with Coomassie blue, a discrete pattern of bands is observed compatible with the simultaneous expression of several VHHs. Since the different VHHs have similar sizes, they were then resolved based on isoelectric point differences by two-dimensional electrophoresis. Thus, 25 μg of the affinity purified recombinant polyclonal mixture was resolved in 2D gels with a pH range between 4.0 and 9.0, and subsequently detected by silver staining. FIGURE 2B shows the result of 2D electrophoresis, in which it is possible to identify a minimum of 9 differentiable spots in the band of molecular weights corresponding to VHH type proteins, which demonstrates the oligoclonal composition of the sample.

EXAMPLE 2. Production of a polyclonal mixture of VHH antibodies

This example demonstrates the possibility of producing complex or very complex repertoires (N> 100) of recombinant proteins of unknown composition in reproducible form. For this, a Goldengate reaction was performed with the VHHs amplified with the SEQ oligos. ID. No. 1, and SEQ. ID. No. 2 from cDNA obtained from a fraction of camel blood nucleated cells and said reaction mixture was directly transformed into competent Agrobacterium GV201 1 cells subsequently seeded in LBAgar plates supplemented with 50 μg / mL kanamycin. The resulting 2x10 ⁴ clones were collected directly from the plates in 0.5 mL aliquots of LB medium and frozen at -80 ° C in the presence of 15% glycerol. Subsequently, one of the aliquots was thawed and subcultured in 5 mL of LB for 2 hours. The resulting culture (pGTMV_VHHp culture) was centrifuged at 500 g for 15 minutes and the cell pellet was resuspended in 10 mL of infiltration buffer (10mM MES pH 5.6, 10mM calcium chloride, 2μΜ acetosyringone). Similarly, we proceeded with Agrobacterium cultures that house the 5 ^' deconstructed TMV module (pICH 17388) and the integrase module (PICH 14011) necessary for the establishment of the viral replicon (Marillonnet et al., 2004b). The OD of each Agrobacterium culture was measured and the relevant dilutions were made in infiltration buffer to bring each culture to an OD = 0.1 measured in a spectrometer. Subsequently, the cultures were combined in the appropriate ratio (1: 1: 1) (pGTMV_ VHHp: plCH17388: PICH1401 1) and the mixtures were infiltrated in N. benthamiana leaves. To verify the reproducibility of the polyclonal expression, the same experiment was repeated on two other occasions with a different group of plants based on two new cryopreserved aliquots of the pGTMV_VHHp. In total, three independent experiments (EXP1, EXP2 and EXP3) were performed. In all cases, they were allowed six days after infiltration and collection. After 6 dpi in each case, the plant material was collected separately and the recombinant proteins were purified following the procedure described in the previous experiment. A PAGE analysis of the purified VHHs revealed no differences in the size pattern of the mixture (FIGURE 3A). This figure shows the runs corresponding to the last wash (F) and two successive elutions (E1 and E2) of the purification process of one of the experiments. In said electrophoresis, revealed with Coomassie blue, a continuous pattern of bands is observed compatible with the simultaneous expression of multiple VHHs.

Subsequently, the three polyclonal mixtures of VHHs were solved together in response to isoelectric point differences by two-dimensional electrophoresis. Thus, 10 μg of each affinity purified recombinant polyclonal mixture were each labeled with a different fluorophore and subjected to a DIGE analysis resolving on a 2D gel with a pH range between 4.0 and 9.0. Subsequent scanning shows the 2D footprint of each of the recombinant mixtures labeled with a different fluorophore. The distribution pattern of the spots was very similar in the three experiments (FIGURE 3B), which demonstrates the reproducibility of the technique. 2D gels were analyzed with the Decyder software package. A minimum number of 216 major spots were detected. All major spots turned out to be coincident in all three experiments. From the volume data of each spot, a dispersion analysis of the data between the different experiments taken two by two was performed. The narrow distribution of the dispersion between experiments is shown graphically in FIGURE 4A, indicating high reproducibility between independent experiments. The scatter plot shown in FIGURE 4 represents for each point the logarithm of the ratio between the relative intensities of that point in the two experiments in comparison. The height on the ordinate axis represents the intensity (volume) of each spot analyzed. It is observed that only some spots of very low intensity have significant differences, possibly due to differences in sensitivity, while most of the spots are centered on a very narrow margin around the zero logarithm value of the expression ratios. Mass sequencing assays have been performed that show the production of complex repertoires of N recombinant molecules. For this, samples of leaves corresponding to the EXP1 and EXP2 experiments mentioned above (1.5 grams of plant material per each experiment) were taken and the total RNA was extracted from these tissues. After DNAse treatment, DNA synthesis was copied using a specific oligonucleotide shown as SEQ. ID. No. 13. The resulting samples were amplified by PCR in 25 cycles using a high fidelity polymerase (Platinum PCR Super Mix Hihg Fidelity, Life technologies). For this, an oligonucleotide whose sequence is shown as SEQ was used as the direct primer. ID. No. 14, and as reverse oligonucleotide reverse primers whose sequences are shown as SEQ. ID. No. 15 for sample EXP1 and SEQ. ID. No. 16 for the EXP2 sample respectively. Each oligonucleotide incorporates a barcode that allows the identification of the sample to which it belongs during the massive sequencing process. The amplified fragments correspond to the variable region of the VHH antibodies. The sequencing of the hypervariable domain CDR3, located within said region allows obtaining a good estimate of the diversity of the VHH sequence repertoire expressed in the plant. The resulting amplicons were purified and brought to a final concentration of 50 ng ^ L. Ten microliters of each sample were used in mass sequencing using Ion Torrent technology. In total, from the EXP1 sample 1105958 readings were obtained and from the EXP2 sample 148815 readings were obtained. The resulting sequences were analyzed and assembled with the iAssembler program to generate a collection of 150 nucleotide unigenes, which correspond approximately to the different CDR3 regions expressed in the plant (http://bioinfo.bti.cornell.edu/tool/iAssembler /). The number of readings for each unique served as an estimate of the frequency of expression of each CDR3. FIGURE 5 shows the frequency distribution for the most abundant CDRs in both experiments. As can be seen, despite the extreme diversity of the repertoire of CDR3 regions expressed in the plant, the relative proportion of each CDR3 remained constant with very slight variations in the two independent experiments. It can therefore be concluded that the analysis of the repertoire of transcripts corresponding to the CDR3 regions expressed in the multitransgenic plants shows a high complexity (more than 2000 variants expressed simultaneously represented by two or more identical readings), despite which the composition of the The resulting mixture is highly stable. The results of experiments thus support the data obtained by electrophoresis of proteins in two dimensions. Finally, the production of complex repertoires of recombinant antibodies obtained from hyper-immunized animals has been tested. For this, three blood samples (C1 H, C2H and C3H) were obtained from three hyperimmunized camels in front of a cocktail of three snake venoms (Crotalus simus, Crotalus scutulatum and Bothros θε βή using a standard hyperimmunization protocol. Blood samples of the same specimens obtained prior to immunization were also available (C1 P, C2P and C3P preimmune samples respectively). From each and every sample libraries were generated in Agrobacterium following the procedure described above: Goldengate reaction with the VHHs amplified with oligos SEQ ID No. 1, and SEQ ID No. 2 from cDNA obtained from a fraction of nucleated camel blood cells and said reaction mixture was directly transformed into cells competent Agrobacterium GV201 1 sowing later on LBAgar plates The 2x10 ⁴ clones resulting from each library were collected directly from the s plates in 0.5 mL aliquots of LB medium and frozen at -80 ° C in the presence of 15% glycerol. An aliquot of each library was combined with the Agrobacterium pICH 17388 and pICH 14011 cultures in the appropriate ratio (1: 1: 1) and agrofiltered separately on Nicotiana benthamiana leaves following the procedure described above. In this way six different somatic mosaics (multitransgenic plants) called C1 H, C2H C3H, C1 P, C2P and C3P respectively were obtained. Six days after the agroinfiltration, the samples (whole leaves) were crushed in a mortar in the presence of liquid nitrogen with PBS buffer in a 1: 3 ratio (tissue weight: buffer volume). The resulting extracts were centrifuged at 10 krpm for 15 min at 4 ° C and the supernatants were passed through Ni Agarose affinity columns in order to purify the repertoire of VHHs contained in each extract. Next, the activity of binding each repertoire to different snake venoms immobilized on ELISA plates was determined. For this, the wells of the plates were upholstered with lyophilized poisons and resuspended in carbonate buffer at a concentration of 2 μg / mL. Subsequently, the wells were incubated with serial dilutions of the different purified VHH repertoires and then incubated with a rabbit polyclonal serum anti-VHHs. Finally, a secondary anti-rabbit antibody conjugated to peroxidase was used to reveal the binding. In all cases, the repertoires obtained from plants expressing hyperimmunized libraries (C1 H, C2H and C3H) showed a high binding capacity against the components of the poisons used in immunization, while the anti-venom activity of preimmune libraries (C1 P, C2P and C3P) showed no activity. FIGURE 6A shows as an example the in vitro activity against the C. simus venom (using the immunization cocktail) of the three preimmune samples (raw extracts), compared to the activity of the equivalent pre-immune samples. In FIGURE 6B it is observed that the anti-poison activity is highly specific, since the repertoires C1 H, C2H and C3H react strongly against the venom of C. simus, but on the contrary they have no activity against bovine albumin (BSA ) or against unrelated poisons such as cobra poisons (Naja ninubiae and Naja mossambica). Therefore it can be concluded that by the method of the invention it is possible to produce multitransgenic plants capable of reproducing, at least partially and in a functional manner, the complex immune response of a camelid against a certain antigen. The multitransgenic plants thus obtained express in the form of somatic mosaics and simultaneously and reproducibly, complex or very complex repertoires of immunoglobulins against said antigen.

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Claims

1. Method for the production of at least one complex repertoire of N recombinant molecules, characterized by comprising the following steps: i) assembling a collection of N distinct nucleotide sequences in a viral vector of destination such that a collection of N is generated viral clones; ii) transfer of the collection of viral clones jointly and simultaneously to a host plant, iii) generation of a transiently multi-transgenic plant structured in the form of a somatic mosaic where more than 80% of the cells express exclusively one of the N molecules recombinants, or express it mostly, in a molar proportion that accounts for at least 80% of the total of N recombinant molecules produced in that same cell.

2. The method according to claim 1, characterized in that the N recombinant molecules are N recombinant proteins.

3. The method according to claims 1-2, characterized in that the viral vector of destination of step ii) is in the form of an infective clone inserted in a binary vector and uses the transient transformation based on Agrobacterium tumefaciens as an input mechanism in Host plant cells.

4. The method according to claim 3, characterized in that the viral vector is devoid of its systemic movement function.

5. The method according to claim 4, characterized in that the viral vector is delivered to the host plant cells by a mechanical system.

6. The method according to claim 5, characterized in that the mechanical system is selected from the following group: vacuum infiltration, overpressure infiltration and spray.

7. The method according to any of claims 1-6, characterized in that the N nucleotide sequences of step i) encode at least one sequence variant of a family of proteins.

8. The method according to claim 6, characterized in that the family of proteins is selected from the following group: enzymes, immunoglobulins, membrane receptors, intracellular receptors, and lectins.

9. The method according to any of claims 1-8, combined with an expression system without homologous interference.

10. The method according to any of claims 1-9, characterized in that the N nucleotide sequences of step i) encode at least one variable sequence of at least one immunoglobulin.

The method according to claim 10, characterized in that the immunoglobulin is produced by lymphocytes obtained from animals immunized against an antigen.

12. The method according to any of claims 1-9, characterized in that the N nucleotide sequences of step i) encode at least one variable sequence of at least one immunoglobulin or its fragment, selected by an in vitro selection technique of recombinant antibodies .

13. Use of the method described in any of claims 1-12, to recombinantly produce a complex mixture of homopolymers.

14. Use of the method described in any of claims 1-12, to recombinantly produce a complex mixture of heteropolymers.

15. A transiently multi-transgenic plant or a fragment thereof, characterized in that it is obtained in step iii) of the method described in any of claims 1-12.

16. The plant or fragment thereof according to claim 15, characterized in that it expresses at least one complex repertoire of polyclonal antibodies.

17. The plant or fragment thereof according to claim 16, characterized in that the polyclonal antibodies are similar to those produced by an animal or a group of animals in response to an immunization process against an antigen, a group of antigens or a pathogen.

18. A crude extract obtained from a plant described in any of claims 15-17.

19. A purified or partially purified mixture of antibodies obtained from an extract described in claim 18.

20. A complex repertoire of N recombinant molecules characterized in that it is produced by the method described in any of claims 1-12.

21. The repertoire according to claim 20, characterized in that the N recombinant molecules are N recombinant proteins.

22. The repertoire according to claim 21, characterized in that the N recombinant proteins are selected from the following group: enzymes, immunoglobulins, membrane receptors, intracellular receptors, lectins, polyclonal antibodies, antivenoms based on antisera, immunological sera for passive immunity , and intravenous immunoglobulins.