METHOD FOR MANUFACTURING TRANSGENIC PLANT PRODUCING IMMUNOGENIC COMPLEX PROTEINS AND IMMUNOGENIC COMPLEX PROTEINS OBTAINED THEREFROM

Abstract

The present invention relates to a method for manufacturing a transgenic plant producing immunogenic complex proteins and immunogenic complex proteins obtained therefrom and, more specifically, to a method for manufacturing a transgenic plant producing immunogenic complex proteins, a plant manufactured by the method, and immunogenic complex proteins obtained from the plant, wherein the method comprises the steps of: (a) manufacturing a transgenic plant expressing an antigen; (b) manufacturing a transgenic plant expressing an antibody specific to the antigen in step (a); and (c) cross-breeding the plants in steps (a) and (b) to manufacture a cross-bred plant. Immunogenic complex proteins can be mass-produced through the method for manufacturing a transgenic plant, comprising steps (a) to (c), and the transgenic plant manufactured by the method, of the present invention. Further, the immunogenic complex proteins (antigen-antibody complex) obtained from the plant have a gigantic four-dimensional structure, thereby having an excellent immune reaction boosting effect, thus exhibiting an excellent antibody producing capacity in a host animal, even without the use of an immune adjuvant.

Claims

1. A method for preparing a transformed plant producing an immunogenic complex protein, the method comprising: (a) preparing a transformed plant expressing an antigen; (b) preparing a transformed plant expressing an antibody specific to the antigen in step (a); and (c) mating the plants in steps (a) and (b) to prepare a mated plant.

2. The method of claim 1, wherein the antigen is a chimeric antigen comprising (i) and (ii) below: (i) an immune response domain comprising an antigenic protein; and (ii) a target binding domain comprising an Fc antibody fragment.

3. The method of claim 2, wherein the target binding domain (ii) further comprises a hinge region of an immunoglobulin, a heavy chain CH1 domain, or a linker.

4. The method of claim 1, wherein the antigen in step (a) is a colorectal cancer cell surface protein GA733.

5. The method of claim 2, wherein the Fc antibody fragment (ii) comprises a hinge region of IgG specific to GA733, a CH2 domain, and a CH3 domain.

6. The method of claim 1, wherein the antigen in step (a) is GA733-FcK chimeric antigen represented by SEQ ID NO: 9.

7. The method of claim 1, wherein the antibody in step (b) is a monoclonal antibody.

8. The method of claim 1, wherein the antibody in step (b) is a bivalent antibody.

9. The method of claim 1, wherein the antibody in step (b) is an antibody specific to GA733-FcK chimeric antigen, the antibody comprising a heavy chain represented by SEQ ID NO: 12 and a light chain represented by SEQ ID NO: 13.

10. The method of claim 1, wherein the antigen in step (a) and the antibody in step (b) comprise an endoplasmic reticulum retention signal sequence.

11. The method of claim 1, wherein the plant is Nicotiana tabacum.

12. A plant producing an immunogenic complex protein, the plant being prepared by the method of claim 1.

13. The method of claim 12, wherein the immunogenic complex protein is an antigen-antibody complex of GA733-FcK chimeric antigen and an antibody specific thereto.

14. An immunogenic complex protein obtained from the plant of claim 12.

15. The method of claim 14, wherein the immunogenic complex protein is at least one selected from the group consisting of a chimeric antigen-antibody monomolecular form, a pentameric form obtained by polymerizing the chimeric antigen-antibody monomolecular monomers, and a linear structure obtained by cross-linking the chimeric antigen and antibody.

16. A vaccine composition comprising the immunogenic complex protein of claim 14 and a pharmaceutically acceptable carrier or diluent.

17. The immunogenic complex protein of claim 14 for preparing vaccine.

18. A method for immunization, the method comprising administering an effective amount of the immunogenic complex protein of claim 14 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0157] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0158] FIG. 1 depicts chimeric antigen, and specifically, illustrates a structure of colorectal cancer cell surface specific protein-Fc (GA733-FcK).

[0159] FIG. 2 depicts bivalent, monospecific antibody against antigen, and specifically, illustrates a structure of colorectal cancer cell surface specific protein-Fc-specific antibody (CO17-1AK)

[0160] FIG. 3 is a schematic diagram of a procedure obtaining a T1-generation plant through cross-pollination of plants expressing colorectal cancer cell surface specific protein-Fc (GA733-FcK) and colorectal cancer cell surface specific protein-Fc-specific antibody (CO17-1AK), respectively.

[0161] FIG. 4 illustrates results of selecting plants having two genes (GA733-FcK and CO17-1AK) out of T1-generation plants (NOs. 1 to 13) using PCR (GA: standard GA733-FcK, CO: standard mAb CO17-1AK, NT: Non-Transgenic plant, HC: heavy chain of CO17-1AK, LC: light chain of CO17-1AK).

[0162] FIGS. 5A and 5B illustrate western blot results of expression of GA733-FcK gene (FIG. 5A) and CO17-1AK gene (FIG. 5B) in plant NOs 3, 4, 6, 9, and 11.

[0163] FIG. 6 illustrates the results of investigating, using SDS-PAGE, whether two proteins (GA733-FcK and CO17-1AK) were purified in T1-generation plant NO. 4.

[0164] FIG. 7 illustrates the results investigating, using two-color western blot, whether two proteins (GA733-FcK and CO17-1AK) were simultaneously expressed in protein samples purified from T1-generation plant NO. 4.

[0165] FIG. 8a is a schematic diagram showing the binding of capture antibody and antigen (chimeric antigen in the present invention, specifically, GA733-FcK protein) and the binding type of detection antibody recognizing the binding antigen-antibody complex, in sandwich ELISA (capture antibody: green, detection antibody: blue).

[0166] FIG. 8b illustrates the comparative results of binding signals of capture antibody and protein, in sandwich ELISA, when different protein samples (GA.sup.P, GA.sup.P+CO.sup.P, GA.sup.PCO.sup.P) were treated on the same capture antibody (CO.sup.M or CO.sup.P).

[0167] FIG. 9a illustrates the SPR measurement results when GA.sup.P-fixed chip was treated with CO.sup.M, CO.sup.P, GA.sup.P+CO.sup.P, and GA.sup.PCO.sup.P samples.

[0168] FIG. 9b illustrates the SPR measurement results when CO.sup.P-fixed chip was treated with GA.sup.M, GA.sup.P, GA.sup.P+CO.sup.P, and GA.sup.PCO.sup.P samples.

[0169] FIGS. 10a-10f exemplify complex structures showing a linear form, out of immunogenic complex proteins expressed in the T1-generation plant of the present invention. Specifically:

[0170] FIG. 10a shows the simplest type of chimeric antigen-antibody dimeric form, out of chimeric antigen-antibody complexes expressed in T1-generation plant.

[0171] FIG. 10b shows an example of a linear form of chimeric antigen-antibody complex, out of chimeric antigen-antibody complexes expressed in T1-generation plant.

[0172] FIG. 10c shows, as an example of a fusion protein expressed in T1-generation plant, a structure of a fusion protein called Fab arm exchanged fusion protein herein.

[0173] FIG. 10d shows a protein dimeric form by the Fab arm exchanged fusion protein.

[0174] FIG. 10e shows an example of a linear form of complex, out of protein complexes by the Fab arm exchanged fusion protein.

[0175] FIG. 10f shows another example of a linear form of complex, out of protein complexes by the Fab arm exchanged fusion protein.

[0176] FIGS. 11a and 11b exemplify complex structures showing a circular form, out of immunogenic complex proteins expressed in the T1-generation plant of the present invention. Specifically:

[0177] FIG. 11a shows an example of a pentamer structure, out of circular polymerization types of chimeric antigen-antibody monomolecules expressed in T1-generation plant.

[0178] FIG. 11b shows another example of a pentamer structure, out of circular polymerization types of chimeric antigen-antibody monomolecules expressed in T1-generation plant.

[0179] FIG. 12 illustrates electron microscopic observation images of a structure of a protein sample obtained in the parent-generation plant transformed to express GA733-FcK (chimeric antigen). On each image, scale bar expressed by a white horizontal bar indicates 10 nm.

[0180] FIG. 13 illustrates electron microscopic observation images of a structure of a protein sample obtained in T1-generation plant. On each image, scale bar expressed by a white horizontal bar indicates 10 nm.

[0181] FIG. 14 illustrates SPR confirmation results of vaccination effect (antibody production effect in serum) after each protein sample was injected into mice without an immune adjuvant.

[0182] FIG. 15 illustrates the confirmation results of interleukin-4 (IL-4) production in mice vaccinated with respective proteins.

[0183] FIG. 16 illustrates the confirmation results of interleukin-10 (IL-10) production in mice vaccinated with respective proteins.

[0184] FIG. 17 illustrates the confirmation of activity of anti-colorectal cancer antibody in serum obtained from mice administered with respective vaccine candidate materials, showing the size of colorectal cancer over time.

[0185] FIG. 18 illustrates mass analysis results of the sugar structure obtained by purifying GA.sup.P(GA733-FcK), CO.sup.P(CO17-1AK), and GA.sup.PCO.sup.P (GA733-FcK CO17-1AK) in plant, confirming that GA.sup.PCO.sup.P obtained through cross-pollination had the similar sugar structure pattern to its parent-generation plant.

MODE FOR CARRYING OUT THE INVENTION

[0186] Hereinafter, the present invention will be described in detail.

[0187] However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

[0188] Preparation of Antigen-Expressing Transformed (or Transgenic) Plant and Antibody-Expressing Transformed (or Transgenic) Plant

[0189] Colorectal cancer cell surface specific protein-Fc (GA733-FcK antigen) was prepared by the same method as described in Korean Patent NO. 10-1054851 by the present inventors, and the literature by Zhe Lu et al.

[0190] Briefly speaking, the genes encoding the colorectal cancer cell surface specific protein GA733 (SEQ ID NO: 1) modified by N-terminal extension with a 30-aa plant ER signal peptide (SEQ ID NO: 3) and the human IgG1 Fc sequence (SEQ ID NO: 6) with ER retention signal (SEQ ID NO: 8) added at the IgG Fc C-terminal were disposed to arrange a gene sequence (see SEQ ID NO: 10) to express GA733-FcK recombinant fusion protein (SEQ ID NO: 9). An expression cassette was constructed by disposing a cauliflower mosaic virus (CaMV) 35S promoter and a tobacco etch viral 5-leader sequence (TEV) in front of the GA733-FcK gene. The constructed colorectal cancer cell surface specific protein-Fc expression cassette as such was inserted into the pBINPlus vector using restriction enzyme HindIII to prepare a plant expression vector.

[0191] In order to express, in a plant, mAb CO17-1A (heavy chain: SEQ ID NO: 11, light chain: SEQ ID NO: 13), known as an antibody against the colorectal cancer cell surface specific protein (GA733), the gene sequence of the ER retention signal was added to the C-terminal of the IgG heavy chain of the mAb CO17-1A, which was named mAb CO17-1AK (heavy chain: SEQ ID NO: 12, light chain: SEQ ID NO: 13). The gene sequences encoding heavy and light chains of mAb CO17-1AK were inserted into pBI121 plant expression vector. The cauliflower mosaic virus (CaMV) 35S promoter and alfalfa mosaic virus untranslated leader sequence (AMV) were inserted to be disposed in front of the heavy chain gene. In addition, the potato proteinase inhibitor II promoter (Pin2p) was inserted in front of the light chain gene to construct an expression cassette. The constructed heavy chain and light chain expression cassettes as such were treated with restriction enzymes HindIII and EcoRI, followed by its insertion into the plant expression vector pBI121.

[0192] The prepared plant expression vectors were introduced into Agrobacterium tumefaciens using electroporation, respectively. Agrobacterium retaining the inserted genes were then selected and cultured. The cultured Agrobacterium was inserted into the young leaves after formation of a cut with a size of 1-3 cm. The plant leaves were then transferred to solid plant medium, and then cultured on Murashige and Skoog solid medium (Dachfu, Haarlem, Netherland) supplemented with hormones, such as NAA (acetic acid) and BA (6-benzyl-amino-purine), and kanamycin (100 mg/L) until callus was generated. New transformant plants were generated 3-4 weeks after the culture.

Example 2

[0193] Mating of Antigen-Expressing Plant and Antibody-Expressing Plant and Screening First-Generation Plant Simultaneously Expressing Traits of Parent-Generation

[0194] The cross-pollination was performed (see FIG. 3) by artificially placing the stamen of the colorectal cancer cell surface specific protein-Fc antibody (mAb CO17-1AK antibody) onto a flower bud of the plant expressing colorectal cancer cell surface specific protein-Fc (GA733-FcK antigen) produced from <Example 1>. The seeds obtained through the cross-pollination were germinated at 23 C. to grow a plant, thereby obtaining a total of 13 T1-generation (GA733-FcKCO17-1AK) plants. The presence or absence of two genes in the T1-generation plants was confirmed using PCR method. The plants having two genes in each plant subject were selected and screened (see, FIG. 4). A specific experiment method was as follows.

[0195] Genomic DNA was separated and purified using Dneasy kit (Quiagen, Hilden, Germany) from leaves of the plant expressing the colorectal cancer cell surface specific protein-Fc (GA733-FcK antigen), the plant expressing the colorectal cancer cell surface specific protein-Fc antibody (mAb CO17-1AK), and the plant (GA733-FcKCO17-1AK) obtained through the cross-pollination of the above two plants. The plant leaves were taken in approximately 90-100 g, instantly frozen in liquid nitrogen, and then pulverized. After the pulverization, pure plant genomic DNA was purified according to the method recommended by the Dneasy kit manufacturer. PCR was performed using each isolated genomic DNA as a template, a primer of colorectal cancer cell surface specific protein-Fc (GA733-FcK antigen), and primers of heavy chain and light chain of the colorectal cancer cell surface specific protein-Fc antibody (mAb CO17-1AK). The previously isolated genomic DNA (1 l) and iTaq premix (Intron Biotechnol. Inc., Seongnam, Korea) were mixed, and forward primer 5-GTCGACACGGCGACTTTTGCCGCAGCT-3 (SEQ ID NO: 17) and reverse primer 5-GAGTTCATCTTTACCCGGGGACAG-3 (SEQ ID NO: 18) of GA733-FcK were added at 10 pmol/l. PCR conditions were as follows: 30 cycles of denaturation-annealing-elongation at 94 C. for 30 s, 67 C. for 30 s, and 72 C. for 30 s. In the same manner, each PCR was performed using forward primer 5-ATGGAATGGAGCAGAGTCTTT-3 (SEQ ID NO: 19) and reverse primer 5-ATCGATTTTACCCGGAGTCCG-3 (SEQ ID NO: 20) of the heavy chain of mAb CO17-1AK and forward primer 5-ATGGGCATCAAGATCGAATCA-3 (SEQ ID NO: 21) and reverse primer 5-ACACTCATTCCTGTTGAAGCT-3 (SEQ ID NO: 22) of the light chain of CO17-1AK.

[0196] As shown in FIG. 4, the results verified that both of GA733-FcK and CO17-1AK were expressed in plant NOs. 4, 6, and 11.

Example 3

[0197] Verification on Gene Expression in Selected T1-Generation Plants

[0198] The expression of antigen and antibody for the plants selected in <Example 2> was investigated.

[0199] <3-1> Western Blot

[0200] 100 mg of fresh leaves were taken from each of the transformed (or transgenic) plants GA733-FcK and CO17-1AK in <Example 1> and GA733-FcKCO17-1AK (T1-generation plants) in <Example 2>, and put in 300 l of 1PBS KCl, Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4), followed by sufficient pulverization. The supernatant of the pulverized leaves was subjected to electrophoresis on 10% SDS-PAGE gel. The supernatant was transferred to a nitrocellulose membrane, and then blocked with 5% skim milk (Fluka, Buchs, Switzerland) at 4 C. for 16 h. For secondary antibody treatment, anti-EpCAM/TROP1 (R&D system, Minneapolis, Minn.) and anti-mouse IgG H+L (Bethyl, Montgomerty, Tex.) diluted at a ratio of 1:5,000 were treated. Membrane washing was performed using 1PBS (Tween 0.1%) buffer three times for 10 min each time. After the buffer was removed from the membrane, the membrane was reacted with Supersignal chemiluminescence substrate (Thermo, Fisher Scientific, Roskilde, Rosilde, Denmark), and then photosensitized on the X-ray film.

[0201] The western blot test verified that both of the antigen (FIG. 5a) and antibody (FIG. 5b) had high expression rates in T1-generation plants NOs. 4, 6, and 11.

[0202] <3-2> Electrophoresis and Two-Color Western Blot

[0203] Out of the plants confirmed to have expressed the antigen and the antibody in Example <3-1>, plant NO. 4 was grown in vivo condition (greenhouse). The leaves of the transformed (or transgenic) plants were purified, and then its protein molecular size was confirmed, while the plants expressing the two genes were confirmed through the two-color western blot. A specific experiment method was as follows.

[0204] Plant lines 4, 6, and 11 confirmed in vitro conditions were planted in the nursery bed soil (Sunshine Mix5, Agawam, Mass.). The temperature and humidity of the green house were 34 C. and 64% RH which are the average condition during July to September. When the plants were grown to adult plants and produced flowers, only leaves were collected and harvested, and then stored at 70 C. The collected leaves were used to purify antigen-antibody proteins. The plant purification was performed using protein G column (GE healthcare, Little Chalfont, United Kingdom). In each sample, GA.sup.M is the chimeric antigen protein produced by the same method as described in <Example 1> using the GA733 protein and Anti-Human EpCAM/TROP1 MAb [Clone 158210] (Mouse IgG2A, CATALOG# MAB960) purchased from the R&D systems, and CO.sup.M means mouse-derived mAb.sup.M CO17-1A. GA.sup.P(GA733.sup.P-FcK), CO.sup.P(mAb.sup.P CO17-1AK), and GA.sup.PCO.sup.P(GA733.sup.P-FcKmAb.sup.PCO17-1AK) were the plants expressing a chimeric antigen and an antibody against the same, and a recombinant protein obtained from the plant prepared through cross-pollination of the plants as described in Examples 1 & 2. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was made to 10% gel, and the respective protein samples were subjected to electrophoresis.

[0205] For the two-color western blot, 8 ul of the respective purified samples GA.sup.M (chimeric antigen of GA733 and anti-Human EpCAM/TROP1 MAb), GA.sup.P (GA733.sup.P-FcK), CO.sup.M (mAb.sup.M CO17-1A), CO.sup.P (mAb.sup.P CO17-1AK), GA.sup.PCO.sup.P (GA733.sup.P-FcKmAb.sup.P CO17-1AK) at a concentration of 0.5 g/l were mixed with 2 l of 5 loading buffer. Electrophoresis was performed using 10% SDS-PAGE, and the membrane was transferred to a nitrocellulose membrane, and then blocked with 5% skim milk (Fluka, Buchs, Switzerland) buffer at 4 C. for 16 h. For secondary antibody treatment, goat anti-human IRDye 800 CW (LI-COR, Lincoln, Nebr.) and goat anti-mouse IRDye 680 LT (LI-COR, Lincoln, Nebr.) were mixed with skim milk at a ratio of 1:15,000, followed by treatment at room temperature for 16 h. Membrane washing was performed using 1PBS (Tween 0.1%) buffer three times for 10 min each time. The buffer of the membrane was removed, and then protein bands were confirmed using the infrared imaging system Odyssey detector (LI-COR, Lincoln, Nebr.).

[0206] The results verified that two proteins GA733-FcK and CO17-1AK were purified in T1-generation plants using SDA-PAGE (see FIG. 6). In addition, it was verified through the two-color western blot that two proteins GA733-FcK and CO17-1AK were simultaneously expressed in samples purified from the T1-generation plants (see FIG. 7).

Example 4

[0207] Confirmation of Morphology and Structure of Proteins

[0208] <4-1> Prediction of Structure of Protein Complex Through Sandwich ELISA

[0209] The sandwich ELISA was performed using the samples purified in Example <3-2>.

[0210] Specifically, 100 l of CO.sup.M(mAb.sup.M CO17-1A) or CO.sup.P(mAb.sup.P CO17-1AK) as a capture antibody was dispensed at a concentration of 5 ng/l in each well of the 96-well plat, and cultured at 4 C. overnight. In order to remove non-binding antibodies, the treated solution was removed from the well, and then the plate wells were washed three times with 1PBS. In addition, 150 l of 3% BSA solution was dispensed at 4 C. overnight. After the treated 3% BSA was removed, the wells were washed three times with 200 l of 1PBS. Antigens GA.sup.P(GA733.sup.P-FcK) and GA.sup.P+CO.sup.P(GA733.sup.P-FcK+mAb.sup.P CO17-AK, purified from the plants, and the same amount of proteins purified from the plants were mixed in vitro), and GA.sup.PCO.sup.P (GA733.sup.P-FcKmAb.sup.P CO17-1AK, protein purified from T1-generation plant NO. 4) was treated at 700 ng, 350 ng, 125 ng, and 62.5 ng on the samples, respectively, followed by incubation at 37 C. for 1 hr. In addition, washing was repeated three times with 1PBS. Anti-human Fc-HRP (Jackson ImmunoReseach Labs, west grove, PA) as a detection antibody and 3% BSA solution at a ratio of 1:10,000, were dispensed in 150 l per each well, followed by incubation at room temperature for 2 h. After the incubation, each well was treated with TMB (3,3, 5,5-tetramethylbenzidine) substrate (KPL, Gaithersburg, Md., USA). In addition, the absorbance was confirmed at 450 nm. The binding of the capture antibody and the antigen (chimeric antigen according to the present invention, specifically GA733-FcK protein) and the binding form of the detection antibody recognizing the bound antigen-antibody complex are shown in FIG. 8a.

[0211] As a result, as shown in FIG. 8b, GA.sup.PCO.sup.P showed higher absorbance than GA.sup.P and GA.sup.P+CO.sup.P. Especially, the absorbance signal of GA.sup.P+CO.sup.P was smaller than that of GA.sup.P, and this result was compared with the fact that GA.sup.PCO.sup.P showed higher absorbance than GA.sup.P, indicating that a large quaternary structure was not generated in GA.sup.P+CO.sup.P. Therefore, it was presumed that the antigen-antibody complex of the proteins purified from the T1-generation transformed (or transgenic) plants (especially, plant NO. 4) configures a stronger complex and forms a larger molecule than the antigen-antibody complex generated by the in vitro artificial binding of the antigen and antibody.

[0212] <4-2> Prediction of Structure of Protein Complex Through Surface Plasmon Resonance (SPR)

[0213] In order to validate that the antigen-antibody complex of the proteins purified from T1-generation transformed (or transgenic) plant (especially, transformed (or transgenic) plant NO, 4) configures a stronger complex and forms a large molecule, SPR was performed using GA or anti-GA antibody-coated SPR chip. Specifically, SPR was performed using ProteOn XPR36 surface instrument (Bio-Rad). GA.sup.M (R&D systems) or CO.sup.M was fixed to GLC sensor chip (Bio-Rad) using amine coupling chemistry according to the protocol provided by the manufacturer. The resonance unit (RU) was about 1,6001,800. The chip stabilization was performed by flowing PBS-T buffer at a flow rate of 100 L/min for 60 s. Each sample (15 g/mL) was allowed to flow on the receptor fixed at pH 6.0 at a flow rate of 50 L/min at 25 C. After each measurement, the surface of the sensor chip was regenerated using phosphoric acid. In all experiments, data were 0 or were adjusted according to the standard channel. The dissociation and rate constant were calculated using Proteon Manager (Bio-Rad).

[0214] As a result, as shown in FIG. 9a, on the GA-coated SPR chip, the kinetic signals of GA.sup.PCO.sup.P and GA.sup.P+CO.sup.P were significantly low compared with those of CO.sup.P and CO.sup.M. Further, as shown in FIG. 9b, on the anti-GA-antibody-coated SPR chip, the signal level of GA.sup.PCO.sup.P was lower than that of GA.sup.P+CO.sup.P. These results support that the antigen-antibody complex configuring a large quaternary structure was generated in the T1-generation plants of the present invention.

[0215] <4-3> Electron Microscopic Observation

[0216] It was predicted from the results of Examples <4-1> and <4-2> that large quaternary structures shown in FIGS. 10 and 11 were generated in the T1-generation plants, and this prediction was confirmed. Specifically, for each of the protein samples obtained from the parent-generation antigen-expressing plant (GA733-FcK antigen) prepared in <Example 1> and offspring T1-generation prepared in <Example 2>, the protein structure and morphology were monitored by staining and electron microscope. Protein samples were incubated at 37 C. for 1 h. After the centrifugation, each sample was re-dispersed in PBS for preparing a specimen of transmission electron microscopy (TEM). The sample solution was loaded on a carbon film-coated TEM grid having hydrophilicity by glow ejection. After 90 s, an excessive sample solution was wiped out with distilled water. For negative staining, 1% uranyl acetate was loaded on a grid for 1 min, and then an excessive staining solution was wiped with filter paper. The samples were photographed by the bio-transmission electron microscope.

[0217] FIG. 12 shows a structure of GA733-FcK protein (antigen) expressed in the parent-generation plant, and FIG. 13 shows electron microscopic results of a structure of a protein sample obtained in T1-generation plant in which GA733-F cK and an antibody CO17-1AK against the same were simultaneously expressed. As shown in FIG. 12, the GA733-FcK protein (antigen) is observed in a Y-shape (15 nm) and various shapes, and an antigen protein existing alone was observed. Also, as shown in FIG. 13, a loop-shaped circular form (20-30 nm) shown in FIG. 11 was observed in the protein sample obtained from the T1-generation plant, while a ball-shaped form of 30 nm or larger and an aggregate of 30 nm or larger were also observed.

[0218] It can be seen from the above results that the antigen and the antibody configure a complex having a large quaternary structure with various shapes in the offspring-generation (A733-FcKCO17-1AK) plant produced through cross-pollination of the plant expressing colorectal cell surface specific protein-Fc (GA733-FcK antigen) and the plant expressing colorectal cell surface specific protein-Fc antibody (mAb CO17-1AK antibody).

Example 5

[0219] Measurement of Vaccine Effect

[0220] <5-1> Measurement of Immunization by Vaccination (Measurement of In Vivo Antibody Production)

[0221] The effect of vaccination was investigated by injecting four protein samples into mice.

[0222] Four protein samples used in the present test were as follows: GA.sup.M (chimeric antigen protein produced by the same method as in <Example 1> using GA733 protein and Anti-Human EpCAM/TROP1 MAb [Clone 158210](Mouse IgG2A, CATALOG#. MAB960) marketed by R&D systems), GA.sup.P (GA733.sup.P-FcK), GA.sup.M+CO.sup.M (obtained by mixing in vitro the same amounts of proteins, a chimeric antigen of GA733 and anti-Human EpCAM/TROP1 MAb and mAb.sup.M CO17-1A), GA.sup.PCO.sup.P (GA733.sup.P-FcKmAb.sup.P CO17-1AK)

[0223] Five mice per each group were used, and the four protein samples were injected without an immune adjuvant. 1PBS was administered into a control group. After the injection of the samples, the serum of each group was obtained, and the amount of antibody produced in serum per each group was checked using a surface plasmon resonance (SPR) method as shown in Example <4-2>. Briefly speaking, for the surface plasmon resonance (SPR), a colorectal cancer candidate protein GA.sup.P (GA733-FcK) was attached to a gold chip, and then 10 l of the serum of each of the vaccinated mice was allowed to flow through the gold chip.

[0224] As a result of checking the difference between groups by measuring the amount of antibody produced in the serum of the mice, as shown in FIG. 14, it was verified that the serum of the mice injected with 1PBS (negative control) showed the lowest signal while GA.sup.PCO.sup.P showed a higher value compared with the other test groups, and thus GA.sup.PCO.sup.P induced a higher immune response than any other vaccine candidate group. It was especially verified that GA.sup.PCO.sup.P, which is the immunogenic complex obtained from the plant of the present invention, showed an excellent immunopotentiating effect, compared with the administration effect of GA.sup.M+CO.sup.M, which is the immune complex prepared in vitro. These results are due to the fact that the antigen-antibody complex produced by plant mating of the present invention configures a complex through stronger binding, compared with the antigen-antibody binding produced when the antibody and the antigen are placed in vitro at the same point in the prior art.

[0225] <5-2> Measurement of Immune Cell Activation (Measurement of Cytokine Production)

[0226] The spleen was extracted from each of the vaccinated mice in Example <5-1>, and disrupted together with media, and then dendritic cells and GA733-FcK as an antigen were co-cultured. The co-cultured flask was cultured at 37 C. for 3 days. After the culture, the activation of IL-4 and IL-10 was measured using FACS. The present test checked whether CD4+ of T cells was activated. CD4+ may be divided into classic Th1/Th2/Th17 responses, while IL-4 and IL-10 are factors included in Th2.

[0227] As a result, as shown in FIGS. 15 and 16, the spleen of the mice injected with GA.sup.PCO.sup.P showed the highest IL-4 and IL-10 cytokine values compared with the mice immunized with 1PBS, GA.sup.M, GA.sup.P, or GA.sup.M+CO.sup.M. These results indicate that T cell activation was increased in the mice injected with GA.sup.PCO.sup.P. These results verified that the large molecule antigen-antibody complex of the present invention increases CD4+, and further influences the formation of antibodies.

[0228] <5-3> Comparison of In Vivo Cancer Growth Inhibitory Effect

[0229] Human colon cancer cells, SW 620 cells (110.sup.6) were intradermally (i.d.) inoculated in the back of 6-week age BALB nu/nu mice (three animals per each group, Japan SLC Inc., Hamamatsu, Shizuoka, Japan) to construct tumor xenograft mouse models. 40 l of the serum obtained from each of BALB/c mice immunized with 1PBS, GA.sup.M, GA.sup.P, GA.sup.M+CO.sup.M, or GA.sup.PCO.sup.P was intraperitoneally injected into six groups of the tumor xenograft mouse models a total of four times every three days (administered at a total of 160 l for 7 days). The positive control group was injected with 100 g of purified mAb CO17-1A(CO.sup.M). The growth of tumor, that is, the tumor volume was recorded on day 8, 10, 12, and 15 after the initial injection of cancer cells, and was calculated on the basis of three main diameters measured by graduated calipers by the following equation: (mm.sup.3)=widthlengthheight.

[0230] The test results are shown in FIG. 17. In nude mice injected with the serum obtained from BALB/c mice xenografted with SW 620 human colon cancer cells and immunized with 1PBS, GA.sup.M, GA.sup.P, GA.sup.M+CO.sup.M, or GA.sup.PCO.sup.P, tumor symptoms appeared on day 8 from the transplantation of cancer cells. Thereafter, the tumor size was abruptly grown in the 1PBS treated group compared with the other test groups. The tumor was significantly quickly grown in the GA.sup.M serum, GA.sup.P serum, and GA.sup.M+CO.sup.M serum administered groups compared with the GA.sup.PCO.sup.P serum or CO.sup.P serum administered group. On day 15, the tumor size of the GA.sup.PCO.sup.P administered group was significantly small compared with those of the other test groups. The tumor growth inhibitory effect in GA.sup.PCO.sup.P administered group was similar to that in the CO.sup.P administered group.

Example 6

[0231] Analysis of Sugar Composition of First-Generation Protein

[0232] For comparison of N-glycan profile among GA.sup.P, CO.sup.P and GA.sup.PCO.sup.P, mass analysis was performed.

[0233] The recombinant protein samples purified from the parent-generation (GA.sup.P, CO.sup.P) and T1-generation (GA.sup.PCO.sup.P) plants were first digested into glycopeptides using pepsin. N-glycans were released from the glycopeptides using PNGas A (Roche), and the released N-glycans were purified using graphitized carbon resin from Carbograph (Alltech). The purified glycans were resuspended in a mixture of 90 L dimethyl sulfoxide (DMSO), 2.7 L of water, and 35 L of iodomethane, and then solid phase permethylation was performed using a spin-column method (Goetz J A et al., 2009). The thus obtained permethylated glycans were mixed in equal volume with 10 mg/mL 2,5-dihydroxybenzoic acid solution (prepared in 1 mM sodium acetate solution). The resulting mixtures were applied onto a matrix-assisted laser-desorption-ionization (MALDI) MSP96 ground steel target plate and dried, followed by MALDI-TOF mass spectrometry. All mass spectra were acquired at a 20 kV accelerating voltage.

[0234] As shown n FIG. 18, the mass analysis results verified that oligomannose glycans (Man 79) were present in all the samples. It was verified that CO.sup.P mainly has a Man 7 glycan structure while GA.sup.P has a Man 79 oligomannoseglycan structure. Like CO.sup.P and GA.sup.P, GA.sup.PCO.sup.P also had an oligomannoseglycan structure. Further, the relative ratio (4:1) of Man 7 and Man 9 in GA.sup.PCO.sup.P was similar to a sum of those in CO.sup.P and GA.sup.P. Therefore, it can be seen that the immune complex expressed in the T1 generation contains almost the same glycan structure as in the proteins of the parent generation.

INDUSTRIAL APPLICABILITY

[0235] As set forth above, the present invention relates to a method for preparing a transformed (or transgenic) plant producing an immunogenic complex protein and an immunogenic complex protein obtained therefrom and, more specifically, to a method for preparing a transformed (or transgenic) plant producing an immunogenic complex protein, the method comprising: (a) preparing a transformed (or transgenic) plant expressing an antigen; (b) preparing a transformed (or transgenic) plant expressing an antibody specific to the antigen in step (a); and (c) mating the plants in steps (a) and (b) to prepare a mated plant, to a plant produced by the method, and to an immunogenic complex protein obtained from the plant.

[0236] Through the method for preparing a transformed (or transgenic) plant, comprising steps (a) to (c), and the transformed (or transgenic) plant produced by the method, immunogenic complex proteins can be mass-produced safely and economically. Furthermore, the immunogenic complex protein (antigen-antibody complex) obtained from the plant has a large quaternary structure, thereby having an excellent effect in boosting immune response, and thus exhibits an remarkable capability in producing antibodies in a host animal even without using an immune adjuvant.