WO2009048354A1

WO2009048354A1 - Method for overproducing anti-her2/neu oncogene antibodies in plant

Info

Publication number: WO2009048354A1
Application number: PCT/RU2008/000638
Authority: WO
Inventors: Yury Leonidovich Dorokhov; Tatyana Valerievna Komarova
Original assignee: Federalnoe Gosudarstvennoe Unitarnoe Predpriyatie 'gosudarstvenny Nauchny Tsentr 'nauchno-Issledovatelsky Institut Organicheskikh Poluproduktov I Krasitelei (Niopik); Institut Fiziko-Khimicheskoi Biologii Im. A.N.Belozerskogo Mgu; BARYSHNIKOV Anatoly Yurievich; KOSORUKOV Vyacheslav Stanislavovich; Kiselev Sergey Lvovich
Priority date: 2007-10-08
Filing date: 2008-10-06
Publication date: 2009-04-16
Also published as: RU2370280C2; RU2007137277A

Abstract

The invention relates to biotechnology, genetic engineering and medicine and provides a method for producing (overproducing) in a plant cell of monoclonal antibodies (mAbs), particularly the anti-Her2/neu-positive cancer cell mAbs that can be used for diagnostics and treatment of breast cancer.

Description

METHOD FOR OVERPRODUCING ANTI-HER2/NEU ONCOGENE

ANTIBODIES IN PLANT

Technical Field

The invention relates to biotechnology, genetic engineering and medicine and can be used for creating plants - overproducers of antibodies, in particular antibodies against HER2/neu-positive cancer cells. Such antibodies can be used for diagnostics and treatment of the breast cancer.

Background Art

The breast cancer affects every ninth female human now and it spreads further with every passing year. Surgical intervention is a treatment at an earlier stage of disease. However perfidy and danger of breast cancer consist in rapid metastasizing which calls for chemo- and radiotherapy. A few years ago, Trastuzumab developed by GENENTECH (US firm) and manufactured by HOFFMAN LA ROCHE (Switzerland) was introduced in medical practice. This drug has been developed on the basis of humanized monoclonal antibodies (mAbs) (huMab4D5-8 or rhuMabHer2), which are produced by the mouse hybridoma line ATCC CRL- 10463 from the American Type Culture Collection. These widespread antibodies present only one of the types of known antibodies interacting with the HER2 oncoprotein (derived from Human Epidermal growth factor Receptor 2). The HER2/neu oncogene is a transmembrane tyrosine proteinkinase ErbB2 having a molecular weight of 185 kDa. The breast cancer patients (about 30%) suffering from the so-called HER2/neu — positive and the most dangerous form of cancer are distinguished for the overproduction of said oncorpotein, which fact causes rapid metastasizing and resistance to a therapeutic treatment. An antibody introduced into a patient's blood flow interacts with an extracellular HER2/neu portion and suppresses division of cancer cells, rarely accompanied by disintegration of the cancer cells. In combination with chemotherapy, the huMab4D5-8 antibodies produce a pronounced therapeutic effect. GENENTECH filed its application for use of mAbs against breast cancer cells SK-BR-3 over-expressing the Her2/neu oncogene for the first time with the US Patent and Trademark Office on August 5, 1994 and received a patent US 5 677 171 on October 14, 1997 (Inventors Hudziak R.M., Shepard H.M., Ullrich A., Fendly B.M.). The GENENTECH claims declare a) creation of the anti- Her2/neu receptor mAbs to inhibit growth of tumor cells; b) mAbs binding an extracellular Her2/neu domain and sensitizing cells to the effect of a cytotoxic factor. During the first 6 months of 1998 applications were filed for clinical use of huMab4D5-8 (Patent US 6 165 464, WO 9704801, WO 9817797). Somewhat later, the use of 454Cl 1 mAbs, produced by the hybridoma HB 8484 was proposed for the purpose by CHIRON CORP. (USA) (June 7, 1995 application), for which patent US 6 054 561 was granted on April 25, 2000.

Humanized huMab4D5-8 mAbs known as Trastuzumab or under a trademark "herceptin" perform particularly well in Russia. It is shown that the use of "herceptin" diminishes the risk of development of remote metastases and increases life span of patients (Ganshina and Lichinitser. 2005. FARMATEKA No 18, 8-11). The cost of treatment of HER2/neu-positive breast cancer using this preparation is very high and amounts to 70 000 USD (Fleck L. "The costs of caring: Who pays? Who profits? Who panders? Hastings Cent Rep 36 (3): 13-17). In general, the cost of production of any target protein in an animal cell culture is high enough and exceeds 10 000 USD per gram protein (Danielli et al., 2001. Trends in Plant Science 6, 219-226). This is several times higher than the cost of production of a similar protein in yeast, insect cells, and even more so in bacteria. Normally mAbs are obtained using an animal cell culture, Chinese hamster ovary cells (CHO) and mouse myeloma cells. The level of mAb production is currently high enough and may reach 3 g of mAb per liter of an animal cell culture (Zhang et al., 2006. Biotechnol Bioeng. 95, 1188- 1197). However, its high cost is composed of expenditures on equipment, a system of maintenance and sterility and also a system of certification and control over media utilizing products of animal origin and increasing the risk of contamination of a mAb preparation with prions and viruses.

Plants have a number of advantages over bacterial, yeast and mammal cells for the production of alien proteins. The plants, just like "factories", a) do not contain human pathogenic viruses and prions and b) do not call for employing expensive equipment (bioreactors), culture medium and sterility systems. The cost of growing original experimental plants is incomparably lower than that of culturing bacterial, yeast and animal cells. The technique of "transient expression" of proteins in plants allows for producing alien proteins in an amount of 10-30% of the total plant soluble protein during a short period of time (5-10 days) when a target gene is incorporated into a binary vector followed by its delivery into cells of a plant to be infected by a method of agroinoculation or agroinfiltration (Kapila et al., 1997. Plant Science 122, 101-108). Under these conditions, the level of accumulation of a target protein is defined by the efficiency of a) binary vector transcription (Rombauts et al., 2003. Plant Physiol. 132, 1162-1176), b) replication (Asurmendi et al., 2004, Proc. Natl. Acad. Sci. USA 101, 1415-1420; Marrillonet et al., 2005. Nature Biotechn. 23, 718-723), c) transcriptome protection from degradation (Voinnet et al., 2003. Plant J. 33, 949-956), d) target protein stability.

Two reports are available in literature concerning the production of a complete anticancer antibody in plants. A small amount of the anti-rectal cancer antibodies (0.02% of the soluble proteins) was accumulated in transgenic tobacco plants (Ko et al.,_2006, Proc. Natl. Acad. Sci. USA 102, 7026-7030). Utilization of viral vectors and a transient expression system provided accumulation of the A5 anti-cancer antibodies in an amount of up to the increment of 3-5% of the soluble protein (Giritch et al., Proc. Natl. Acad. Sci. USA 103, 14701-14706). However, nobody has yet succeeded in expressing the anti- HER2/neu oncogene antibody in plants.

Disclosure of Invention

The present invention relates to a method of production of an antibody in a plant cell, that specifically binds HER2/neu oncoprotein. The method of the present invention comprises a) providing one or more expression vectors directing in a plant cell, the synthesis of antibody heavy/light chains or antigen binding fragments thereof (dsFv, scFv, scFv-Fc); b) introducing said one or more vectors into the plant cell; c) culturing the plant cell under conditions providing for co-expression of said vectors in the cell.

In one of the embodiments, a plant cell is an isolated cell or is in a plant cell culture, plant tissue culture, plant organ culture, in the whole plant or a portion thereof.

In one of the embodiments, a method comprises the use of one or more expression vectors providing the synthesis of a non-amplifiable RNA. In one of the preferred embodiments said one or more expression vectors provide the synthesis of a non- amplifiable RNA under control of an inducible promoter.

In an alternative embodiment, a method comprises using one or more expression vectors which are viral expression vectors. In one of the preferred embodiments, the viral expression vectors used are vectors based on a DNA-containing virus genome. In one more preferred embodiment said viral expression vectors are vectors based on an RNA- containing virus genome. It is most preferable when viral expression vectors based on a DNA/RNA-containing virus genome provide the synthesis of a viral RNA with the aid of an inducible promoter. Preferably the viral expression vectors are selected in such a way so as to preclude mutual competition in the same cell. hi one of the preferred embodiments, an expression vector provides the synthesis of a tobacco mosaic viral RNA encoding an antibody heavy chain. In another preferred embodiment, an expression vector provides the synthesis of a tobacco mosaic viral RNA encoding an antibody light chain.

In the next preferred embodiment, an expression vector provides the synthesis of a potato virus X RNA encoding an antibody heavy chain. In another preferred embodiment, an expression vector provides the synthesis of a potato virus X RNA encoding an antibody light chain.

In still another embodiment, the expression vectors provide the synthesis of antibody light and heavy chains in the same cell. hi the next embodiment, the produced antibody comprises a signal peptide allowing for targeting an antibody to a cell compartment or secretion outside the cell. hi one of the embodiments, an antibody belongs to a class G, or A, or M, or D, or E immunoglobulin. hi still another embodiment, a plant cell is a dicotyledonous or monocotyledonous plant cell, hi a preferable embodiment, the dicotyledonous plant belongs to the Solanaceae family. It is more preferable when the plant of the Solanaceae family is a plant of the genus Nicotiana. The most preferred plant of said genus Nicotiana is N. tabacum or N₁ benthamiana. hi still another embodiment, the dicotyledonous plant is a plant of the Brassicacea, Leguminosae or Chenopodiaceae families. hi the next embodiment, the expression vectors are administered into a plant cell by agroinjection. hi one more of the embodiments, the expression vectors provide stable transformation, hi an alternative embodiment, the expression vectors provide transient transformation. hi its following aspect, the present invention is related to a plant cell producing an antibody interacting with the HER2/neu oncoprotein. In one of the embodiments, the cell is stably transformed with a genetic construct directing antibody synthesis, hi an alternative embodiment, the plant cell is transiently transformed with a genetic construct directing antibody synthesis.

Brief Description of Drawings

Fig. 1. A schematic representation of a binary vector pAl 1866 construct encoding a light chain (L) of a Her2/neu-specific antibody, wherein RB and LB — respectively, the right and left border regions; 35S - cauliflower mosaic virus (CaMV) transcription promoter; PolyA/T - CaMV polyadenylation signal /CaMV terminator of genomic RNA transcription. Sl - leader - 5' - non-translated region of the CaMV genomic RNA.

Fig. 2. Accumulation of an L-chain in plant leaves agroinjected with the vector pA11866, Fig. 1. N₁ benthamiana plant leaves were agroinjected with said pA11866 in the presence of a binary vector encoding a silencing suppressor pl9 of a tomato bushy stunt tombusvirus (lane 1) or the vector pA11691 directing the synthesis of an artificial tRNA in a cell under control of a tRNA promoter (lane 2). Proteins were assayed in a Western blot using the antibodies specific to a Her2/neu-specific human antibody light chain.

Fig. 3. A schematic representation of a construct of the binary vector pA11903 encoding a Her2/neu-specific antibody heavy (H) chain. Symbols are the same as in Fig. 1.

Fig. 4. Accumulation of Her2/neu-specific antibodies in a plant agroinjected with non-amplifiable binary vectors pA11866 and pA11903 and their purification on a protein G-Sepharose column.

Fig. 5. A schematic representation of a construct of the binary vector pA11520 based on a crTMV cDNA and encoding a Her2/neu-specific antibody heavy (H) chain where (left to right) LB - left border region; T - nopalinsynthase terminator; NPT - neomycin phosphotransferase gene; P -nopalinsynthase transcription promoter; Act-2 - Arabidopsis actin transcription promoter 2; crTMV replicase gene; TB - crTMV movement protein; H-chain - heavy chain; NTR - 3' - non-translated crTMV genomic RNA region; T - nopalinsynthase terminator; RB - right border region . Arrows indicate a transcription direction.

Fig. 6. A schematic representation of a construct of the PVX cDNA-based binary vector pA11333 and coding for a Her2/neu-specific antibody light (L) chain, where (left to right) LB - left border region; T - nopalinsynthase terminator; NPT - neomycin phosphotransferase gene; P - nopalinsynthase transcription promoter; 35S - CaMV transcription promoter; PVX RdRp - PVX replicase; L-chain- light chain; NTR- 3' non- translated PVX RNA genomic region; T - CaMV 35S terminator; RB - right border region. Arrows indicate a transcription direction.

Fig. 7. Accumulation of Her2/neu-specific antibodies in a plant agroinjected with the binary vectors pA11520 and pA11333. Fig. 7A. Protein plant extracts 3 (line 1) and 7 (lane 2) days after agroinjection with the vectors pA11520 and pA11333 were fractionated by electrophoresis, the gels were stained with Coomassie. Fig. 7B. Detection of a Her2/neu- specific antibody in a Western blot with the anti-human antibody alpha and kappa chains antibodies. Arrows indicate: Rubisco - chloroplast ribulose bisphosphate carboxylase heavy subunit.

Fig. 8. Accumulation of Her2/neu-specific antibodies in a plant agroinoculated with viral binary vectors pAl 1520 and pAl 1333 and their purification on a Protein G-Sepharose column. Protein bands corresponding to an antibody heavy (H) chain and light (L) chain are marked.

Fig. 9. Antibodies isolated from plants react with an extracellular part of the Her2/neu oncogene. Fig. 9A. Cytological assay of SKBR-3 cells expressing the Her2/neu oncogene and treated by the antibodies isolated from plant. Binding specificity was tested with the aid of rabbit antibodies conjugated with a horseradish peroxidase and reacting with human antibodies. Fig. 9B. — Said SKBR-3 cells after treatment with diagnostic antibodies specifically reacting with the Her2/neu (Dako) (positive control). Fig. 9C and Fig. 9D - Negative controls to A and B, respectively, obtained by treating the SKBR - 3 cells with the rabbit antibodies alone.

Modes for Carrying out the Invention

The invention rests upon the fact discovered by the Applicant that genes coding for mAb heavy and light chains against the Her2/neu oncogene can be expressed in a plant cell to produce a completely functional antibody that specifically binds its "own" antigene. The proposed method contemplates constructing expression vectors directing in the plant cell, the synthesis of an antibody heavy (H) and light (L) chains , insertion of said vectors in a plant cell and culturing the plant cell under conditions providing co-expression in the cell, of said vectors. In general, the method of the instant invention can be used for expression of any anticancer antibodies whose amino - acid sequence is known and published, for example, Bevacizumab (avastin) (Kohen st al., 2007. Oncologist 12, 713-718; patent US 7 241 444), Panitumumab (Easley and Kirkpatrick, 2006. Nature Review 5, 987-988; Patent application USA No 20070179096). A wide variety of the anti-HER2/neu oncogene extracellular domain mAbs are known now for use in treatment of the breast cancer, and the genes encoding same. In particular, the genes coding for the aforesaid antibodies can be recovered from an ICO85 hybridoma synthesizing mouse mAbs against the Her2/neu oncogene (cf. Example 1). mRNA encoding the antibody light and heavy chains can be produced from viral genome-based replication vectors (Example 3) and from non- amplifiable mono- (cf. Example 2) or polycistronic mRNAs using an internal ribosome entry site (Dorokhov et al., 2002. Proc. Natl, Acad. Sci. USA 99 5301-5306). In context of the present invention, the expression vectors encoding the light and heavy chains of the anti-Her2/neu oncogene antibody can be produced on the basis of a binary vector Binl9 (Fig. 1 and Fig. 3 Example 2). These vectors provide the synthesis and accumulation in a plant, of an antibody in an amount sufficient for recovery and purification (Fig. 4 of Example 2).

An advantage of the present invention consists in that it enables one to produce in a plant not only full-length antibodies but also their antigen-binding fragments, for example, dsFv, scFv, scFv-Fc and also camel antibodies which contain heavy chains alone (cf. the review by Jain et al., 2007. Trends in Biotechnology 25, 307-316).

The simultaneous expression of antibody chains in a plant cell with the aid of viral vectors is possible provided that competition between viruses is absent. Our experiments show that TMV and PVX not only do not compete with each other but the PVX even stimulates TMV reproduction. Accumulation of a heavy chain driven by a TMV- vector is increased in the presence of PVX encoding a light antibody chain (Example 3). The analysis of mechanisms of reproduction of sindbis - like phytoviruses shows that they compete at the stage of forming the so-called viral "factories" when a viral replicase interacts with (attaches to) the endoplasmic reticulum membranes. TMV and PVX do not compete with each other at the stage of forming a replication complex and a viral "factory". A turnip yellow mosaic virus (TYMV) forming the viral "fabric" on chloroplast membranes does not compete, either, with the TMV and the PVX and, therefore, can be used for the synthesis in one cell of antibody chains. This approach can be used for selecting other non- competing viral pairs.

The present invention provides for using non-viral vectors for the synthesis of an antibody in a plant cell. These vectors do not compete with each other in a cytoplasm. However, their synthesis and stability in the cytoplasm is controlled by a mechanism of gene "silencing", which fact is likely to lead to lowering a production level of a target antibody. In cases where the production of a target protein is lowered indeed as a result of gene silencing, the present invention comprises utilizing antisilencing proteins. An antisilencing protein can be exemplified by, e.g. the protein P19 of a tomato bushy stunt virus whose gene can be synthesized with ease (Example 2). The same antisilencing effect can be produced by short non-coding RNAs that are synthesized under control of RNA- polymerase III (Fig. 2).

A plant cell that expresses an antibody may be an isolated cell devoid of a cell wall (protoplast) or may be within a cultured explant (tissue, organ) or the whole plant. Culturing cells under artificial conditions is performed in a sterile medium of a known composition and under controlled conditions at a constant temperature of between 26 and 28⁰C and illumination. The present invention contemplates the possibility of using non- coding RNAs to stimulate antibody production in cultured cells both in the form of suspension cultures and plant explants cultured on solid medium or else in the whole plant that is grown in a suitable medium, such as a natural substrate (for example, soil, soil substitutes) or in a hydroponics culture. The antibody can be accumulated within a plant cell or can be exported to culture medium by introducing a signal sequence into its composition. The antibody can be recovered from intracellular contents by disrupting the culture cells or directly from the culture medium in cases where incorporation of the signal sequence in the antibody composition provides secretion to the culture medium.

A DNA coding for an antibody chain can be introduced into a plant cell using methods, an artisan is aware of, e.g. with the aid of electroporation, bombardment with microprojectiles, microinjection and virus infection. Besides, the DNA can be delivered to the plant cell with the aid of Agrobacterium tumefaciens. For the transient expression of an alien gene in leaf tissues, a method of agroinfiltration and agroinoculation is used more frequently (Kapila et al, 1997. Plant Science 122, 101-108). Plant cell transformation can be stable, accompanied by integration of a DNA being introduced into cell genome. A desired effect can likewise be attained by introducing a DNA into a nucleus and transient (temporary) RNA synthesis without stable DNA integration into a host genome (Examples 2 and 3).

A significant and decisive advantage of the present invention is that the synthesis system of the anti-HER2/neu oncogene antibody in a plant can provide a high level production of cheap protein. Out experiments show that 1 kg of N₁ benthamiana leaves is enough for deriving up to 300 - 500 mg of a purified antibody (3-5% of the soluble protein). Chromatography on a Protein G or A column may be helpful in obtaining a pure preparation of the anti-Her2/neu oncogene antibody (Fig. 8), which preparation has the capability to bind an extracellular Her2/neu oncogene domain, as shown in a cytochemical assay (Fig. 9). Our estimations show that the cost of mAbs preparations produced by expression in a plant cell may be 10-20 times lower than that of a similar preparation obtained from CHO animal cells.

Examples

The examples illustrate the most preferred embodiments of the present invention and are given for better explaining the gist of the invention. A person skilled in the art will appreciate that a plurality of modifications can be carried out with regard to usable means and materials and also usable methods without going beyond the scope of the invention.

Example 1. Production of genes encoding heavy and light chains of anti- Her2/neu oncogene mAb.

From a cell culture of ICO85 hybridoma produced in the Laboratory of experimental diagnostics and biotherapy of tumors headed by Prof. A. Yu. Baryshnikov (Research Institute of Diagnostics and Therapy of Tumors, N.N. Blokhin Russian Oncology Scientific Centre, the Russian Academy of Medical Sciences) sequences which code for antibody variable regions with affinity to the Her2/neu oncogene were isolated using genetic engineering methods. The fragments so obtained were incorporated into constructs containing the sequences encoding the class G antibody constant regions.

Example 2. Expression of antibody light and heavy chains against Her2/neu oncogene in plant with the aid of non-viral vector.

The individual genes of antibody light and heavy chains against the Her2/neu oncogene were cloned into a pFF19 subclone containing a 35S-promoter at Ncol and Xhol sites. At the next stage, genes were cloned separately into a vector Bin 19 (Bevan, 1984. Nucleic Acids Res. 12, 8711 - 8721) at Hindlll-EcoRI sites to obtain pA11860 and pA11903 vectors, respectively (Fig. 1 and Fig. 3). Agrobacterium tumefaciens harboring binary vectors expressing a heavy or light chain of the anti-Her2/neu oncogene antibody was inoculated into a liquid 2- YT medium and grown overnight at 28⁰C. The overnight culture was sedimented by centrifugation, 5,000 rpm for 3 minutes. The pellet was then resuspended in an agroinfiltration buffer containing 10 mM MgCl₂ and 10 mM MES, pH 5.0 and administered into a leaf in dilution as required. Fig. 2 shows accumulation of the light chain (L) of the anti-Her2/neu oncogene antibody in N. benthamiana leaves 3 days after agroinjection with the binary vector pA11866. In these experiments, use was made of a construct encoding a tomato bushy stunt virus antisilencing protein P 19 whose gene was synthesized earlier by the instant inventors (Dorokhov et al., 2006. FEBS Letters 580, 3872-3878).

With co-injection of vectors pA11903 and pAH860 into benthamiana leaves, antibody heavy and light chains accumulate therein which can be isolated and purified by affinity chromatography on a Protein G-Sepharose column (Fig. 4).

Example 3. Expression of antibody heavy and light chains against Her2/neu oncogene in plant with the aid of viral vectors.

For producing a TMV genome-based vector (Fig.5), use was made of crTMV cDNA (Dorokhov et al., FEBS Lett. 350, 5-8), in which a replicase gene originated from a closely related strain, a turnip vein clearing virus (Lartey et al., 1994, Arch. Virol., 138, 287-298). A eukaryotic transcription promoter was represented by the Arabidopsis thaliana actin promoter 2 (EMBL AF308778, An et al., 1996, Plant J., 10, 107-121). For producing a PVX genome-based vector (Fig. 6) use was made of cDNA described elsewhere (Komarova et al., 2006, Biochemistry 71, 1043-1049. Agrobacterium tumefaciens harboring binary vectors pA11520 and pA11333 expressing the heavy and light chains of the anti-Her2/neu oncogene antibody, respectively was inoculated into a liquid 2-YT medium and grown overnight at 28⁰C. The overnight culture was sedimented by centrifugation, 5,000 rpm for 3 minutes. A pellet was resuspended in an agroinfiltration buffer containing 10 mM MgCl₂ and 10 nϋvl MES, pH 5.0 and administered into a leaf in dilution as required. Fig. 7 shows accumulation of the anti-Her2/neu oncogene antibody in N₁ benthamiana leaves 4 days after agroinjection with the combination of binary vectors. One can see (Fig. 7) an appreciable accumulation of antibody light and heavy chains that is detected in a gel by Coomassie staining (Fig. 7A) and Western-blot analysis (Fig. 7B). Chromatography on Protein G-Sepharose column is helpful in producing a pure preparation of the anti-Her2/neu oncogene antibody (Fig. 8). Our experiments show that some 300-500 mg of a purified antibody can be derived from 1 kg of N₁ benthamiana leaves.

Example 4. Plant cell synthesized antibody is capable of binding to Her2/neu oncogene on the surface of SKBR-3 cancer cells.

To corroborate functionality of a plant cell synthesized antibody, an immunocytochemical (ICC) assay was used. This assay, alongside with a radioligand and immunoenzyme techniques finds a wide variety of applications in clinical practice. An ICC assay calls for no appreciable time, is conducted rapidly within 2-3 hours and is comprehensively described in literature (cf. Gluzman D.F., Sklyarenko L.M., Nadgornaya V.A., Kryachok LA. Diagnostic immunocytochemistry of tumors. Kiev, MORION; 2003, pp. 28-31). We utilized our antibody in said ICC of SKBR-3 cancer cells harboring the Her2/neu oncogene on their surface. As one can see in Fig. 9 an antibody has the ability to bind a Her2/neu oncogene extracellular domain. Thus, the antibody obtained using the method of the present invention is completely functional and is able to specifically recognize and bind to the Her2/neu oncogene. This opens wide possibilities for use of the method of the present invention to produce anticancer antibodies on a large scale for therapeutical and diagnostic use. The cost price of a diagnostic preparation of antibodies obtained in plants is 12 times lower than that of those commercially available from Dako.

AU patents, publications, scientific papers and other documents and materials, as cited or mentioned hereinbefore, are incorporated herein by reference to an extent as if each and every document was incorporated herein by reference individually or was included here in its complete form.

Specific genes, vectors, plant species, applications, usable materials and methods, as described herein, belong to the preferable embodiments, are cited as examples and are not intended to limit the scope of the invention. On scrutiny of this specification, other subject- matters, aspects and embodiments will come into artisans' mind, that are within the scope of the instant invention. Those skilled in the art will appreciate that various changes and modifications can be performed with regard to the invention, as disclosed herein, without departing from its scope and frames which are defined by the appended claims .

Claims

1. A method for producing an antibody in a plant cell, that specifically binds the Her2/neu oncoprotein, said method comprising: a) providing one or more expression vectors directing in the plant cell, the synthesis of antibody heavy/light chains or antigen-binding fragments thereof (dsFv, scFv, scFv-Fc); b) introducing said one or more vectors into the plant cell; c) culturing the plant cell under conditions providing co-expression of said vectors in the cell.

2. The method according to claim 1, wherein the plant cell is an isolated cell or is within a cultured explant (tissue, organ) or within the whole plant.

3. The method according to claim 1, characterized in that said one or more of expression vectors provide synthesis of a non-amplifiable RNA.

4. The method according to claim 3, characterized in that said one or more of expression vectors provide synthesis of the non-amplifiable RNA with the aid of an inducible promoter.

5. The method according to claim 1, characterized in that said one or more expression vectors are viral expression vectors.

6. The method according to claim 5, characterized in that said viral expression vectors are vectors based on a DNA-containing virus genome.

7. The method according to claim 5, characterized in that said viral expression vectors are vectors based on an RNA-containing virus genome.

8. The method according to any one of claims 5-7, characterized in that said viral expression vectors provide the viral RNA synthesis with the aid of an inducible promoter.

9. The method according to claim 7, characterized in that the expression vector provides the synthesis of a tobacco mosaic virus RNA encoding a heavy antibody chain.

10. The method according to claim 7, characterized in that the expression vector provides the synthesis of a tobacco mosaic virus RNA encoding a light antibody chain.

11. The method according to claim 7, characterized in that the expression vector provides the synthesis of a potato virus X RNA encoding an antibody heavy chain.

12. The method according to claim 7, characterized in that the expression vector provides the synthesis of a potato virus X RNA encoding an antibody light chain.

13. The method according to claim 1, characterized in that the expression vectors provide the antibody light and heavy chains synthesis in the same cell.

14. The method according to claim 5, characterized in that the viral expression vectors are selected in such a way so as to preclude competition with each other in the same cell.

15. The method according to claim 1, characterized in that the antibody contains a signal peptide providing targeting of the antibody to a cell compartment or secretion outside the cell.

16. The method according to claim 1, wherein the antibody belongs to a class G, or A, or M, or D, or E immunoglobulin.

17. The method according to claim 1, characterized in that the plant cell is a monocotyledonous or dicotyledonous plant cell.

18. The method according to claim 17, characterized in that the dicotyledonous plant belongs to the Solanaceae family.

19. The method according to claim 18, characterized in that the Solanaceae family plant is a plant of the genus Nicotiana.

20. The method according to claim 19, characterized in that the genus Nicotiana plant is N₁ tabacum or N₁ benthamiana.

21. The method according to claim 17, characterized in that the dicotyledonous plant is a plant of the Brassicacea, Leguminosae or Chenopodiaceae families.

22. The method according to claim 1, characterized in that said vectors are administered into the plant cell by agroinjection.

23. The method according to claim 1, characterized in that expression vectors that provide stable transformation are administered into the plant cell.

24. The method according to claim 1, characterized in that expression vectors that provide transient transformation are administered into the plant cell.

25. A plant cell as characterized in claim 1, producing an antibody interacting with the Her2/neu oncoprotein.

26. The plant cell according to claim 25, that is stably transformed with a genetic construct directing antibody synthesis.

27. The plant cell according to claim 25, that is transiently transformed with a genetic construct directing antibody synthesis.