WO2005116221A1

WO2005116221A1 - Hctla4-ig fusion protein-coding gene, recombinant vector comprising the same, and process for producing hctla4-ig fusion protein using the same

Info

Publication number: WO2005116221A1
Application number: PCT/KR2005/001582
Authority: WO
Inventors: Sang-Lin Kim; Hyun-Kwang Dan; Wuk-Sang Ryu; Hahn-Sun Jung; Jae-Kyoung Koo; Song-Jae Lee; Mi-Young Park; Cheon-Ik Park; Dong-Il Kim; Sang-Min Lim
Original assignee: Boryung Pharmaceutical Co., Ltd.; Yang, Moon-Sik
Priority date: 2004-05-28
Filing date: 2005-05-27
Publication date: 2005-12-08
Also published as: KR20050112715A; KR100542605B1

Abstract

Provided are a gene coding for a human cytotoxic T lymphocyte antigen 4-Immunoglobulin (hCTLA4-Ig) fusion protein comprising a plant-derived leader sequence, and recombinant vectors pMYN409, pMYN413 and pMYN414, comprising the same. Provided are also host cells transformed with the above-mentioned recombinant vectors, a process for producing a recombinant hCTLA4-Ig fusion protein, involving suspension culturing the host cells, and an hCTLA4-Ig fusion protein prepared using the same. In accordance with the present invention, it is possible to achieve mass production of the recombinant hCTLA4-Ig fusion protein having immunosuppressive activity equal to that of the hCTLA4-Ig fusion protein expressed in conventional animal cells, by use of plant cell suspension culture. Thereby, recombinant proteins can be produced more efficiently and economically, as compared to animal cell culture techniques.

Description

Description HCTLA4-IG FUSION PROTEIN- CODING GENE, RECOMBINANT VECTOR COMPRISING THE SAME, AND PROCESS FOR PRODUCING HCTLA4-IG FUSION PROTEIN USING THE SAME Technical Field

[1] The present disclosure relates to a recombinant vector and transformed cell used in production of a fusion protein. More specifically, the present disclosure relates to a recombinant vector and transformed cell that can be utilized in mass production of a fusion protein having immunosuppressive activity.

[2] Background Art

[3] Generally, immunomodulating substances may be broadly divided into immu- noenhancers and immunosuppressants, depending upon pharmacological action augmenting or suppressing immune functions. Among these, immunosuppressants have received a great deal of attention for their importance in organ transplantation, for example, heart, liver and kidney transplantation because they may serve to prevent transplant rejection. Immunosuppressants have also received a great deal of attention because commercial attention has also been directed to development of drugs for treating autoimmune diseases, which are inflammatory diseases caused by hy- perfunction of the immune system causing the body to attack self-tissue.

[4] T cell activation plays an important role in initiation of transplant rejection. T cells require two types of signals to become fully activated. The first signal is provided by the interaction of the Major Histocompatibility Complex (MHC) of antigen- specific antigen presenting cell (APC) with the T cell antigen receptor (TCR). T cell activation is then initiated by intracellular delivery of the thus -generated activation signal. The second signal is an antigen-nonspecific costimulatory signal. Lack of such a cos- timulatory signal after recognition of antigen by TCR leads to partial activation of T cells or failed T cell activation, which in turn induces T cell anergy that T cell does not react to antigen attack any more. Such T cell anergy is most important for induction of antigen- specific tolerance to prevent transplant rejection. The most important costimulatory signal of T cells is the binding between CD28 and CTLA4 of T cells and the B7 receptors (CD80 and CD86) present on the surface of APCs. CTLA4 has about a 20-fold greater affinity for B7 receptor than CD28 (Linsley et al., J. Exp. Med. 174: 561, 1991; Linsley et al., Immunity, 1:793, 1994). Unlike CD28, binding of CTLA4 to B7 receptor delivers a signal inhibiting or diminishing T cell activation (Sebille et al., Philos. Trans. R. Soc. Lond., B Biol. Sci. 356:649, 2001).

[5] In addition, Linsley et al. (J. Exp. Med., 174: 561, 1991) have reported preparation of a CTLA4-Ig fusion protein in which an Fc portion of immunoglobulin IgG was artificially fused to the C-terminus of CTLA4, and immunosuppressive effects thereof. As a result, an immunoglobulin part of such a CTLA4-Ig makes it possible to achieve effective purification via affinity chromatography, and production of a dimeric CTLA4 protein and a prolonged half- life in vivo. Blockage of the CD28/B7 costimulatory signal by the CTLA4-Ig fusion protein enables prolonged graft survival in animal experimental models including rat cardiac transplantation (Guillot et al., J. Immunol. 164:5228,2000; Hayashi et al., Transpl. Int. 13 (Suppl. 1), S329, 2000; Turka et al., Proc. Natl. Acad. Sci. U.S.A. 89:11102, 1992), mice islet xenograft (Feng et al., Transplantation 67:1607, 1999; Lenschow et al., Science, 257:789, 2000), rat renal transplantation (Tomasoni et al., J. Am. Soc. Nephrol. 11, 747,2000) and monkey islet transplantation (Kirk et al., Proc. Natl. Acad. Sci. U.S.A. 94; 8789,1997; Levisetti et al., J. Immunol. 159: 5187, 1997), and thus suggests therapeutic potential important for practical clinical applications. In fact, clinical trials have shown highly promising results that CTLA4-Ig is therapeutically effective (Abrams et al., J. Clin. Invest. 103: 1243, 1999, J. Exp. Med. 192: 681, 2000; Guinan et al., N. Engl. J. Med., 340:1704, 1999). Immunosuppressants such as conventional commonly used steroid hormone preparations, cyclophosphamide, cyclosporin and FK506 exhibit adverse side effects due to their substantially non-specific suppression of the immune system. Whereas, CTLA4-Ig specifically suppresses only the T cell activation process and thus is expected to exhibit relatively less adverse side effects while having superior immunosuppressive effects.

[6] However, in such a CTLA4-Ig fusion protein, a dosage to humans is up to 10 mg/ kg, each time, that is relatively high as compared to other protein preparations such as cytokines (Abrams, J.R. et al., J. Clin. Invest., 103(9): 1243, 1999: Greene J.L. et al., Arthritis Rheum. 46: 1470, 2002, Rremer, J.M. et al., N. Engl. J. Med., 349(20): 1907, 2003. As such, CTLA4-Ig is extremely costly to produce using conventional animal cell culturing.

[7] Meanwhile, it was recently reported that the CTLA4-Ig having biological activity can be produced from milk of transgenic animals in about 5-fold higher yield than animal cells (Lui V.C.H., et al., J. Immuno., Meth., 277:171, 2003). Further, thanks to advanced plant bioengineering, various efforts and attempts to produce high value- added useful proteins through mass cultivation of plant cells are underway. Due to economical superiority resulting from inexpensive medium components, and easy production, isolation and purification of proteins, such plant cell culture-based production systems have received a great deal of attention as a substitute production system for medicinal proteins such as cytokines, growth factors and im- munomodulators that have been produced by use of conventional microbial or animal cell culture (Miele, L., Trends BiotechnoL, 15: 45-50, 1997; Doran, P.M., Curr. Opin. BiotechnoL, 11: 199-204, 2000). Further, production of recombinant proteins via plant cell culture involves unlike prokaryotic cells such as E. coli, a post-translational modification process similar to that exhibited by animal cells. Thus, it is easy to maintain biological activities of proteins thus prepared and it is also advantageous in view of safety, due to the reduced risk of incorporation of viruses or pathogenic bacteria fetal to humans, as compared to animal cell culture utilizing sera. However, there are still problems that have yet to be solved, such as slow cell growth of plant cell culture systems and lower expression rate of proteins than in microbial or animal cell culture systems. In addition, in order to enter commercialization, there is an urgent need for a cost-effective and efficient large-scale production method. Disclosure of Invention Technical Problem

[8] As a result of extensive and intensive research and study to solve conventional problems, the present inventors have developed vectors and plant cells having a potent promoter system capable of inducing high expression and have discovered that use of such transformed plant cells enables production of highly-concentrated, mass-cultured hCTLA4-Ig fusion protein. Therefore, the present inventors have completed the present invention based on this finding.

[9] Technical Solution

[10] Disclosed herein is a gene coding for a human cytotoxic T lymphocyte antigen 4-Immunoglobulin (hereinafter, also referred to as "hCTLA4-Ig") fusion protein, comprising at least one plant-derived leader sequence selected from the group consisting of rice (Oryza sativa L.), tobacco (Nicotiana tabacum), maize (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), rape (Brassica napus) and potato (Solanum tuberosum).

[11] In this connection, although there is no particular limit to the plant-derived leader sequences so long as they are capable of secreting a recombinant hCTLA4-Ig fusion protein into the outside of plant cells upon suspension culturing, a rice amylase leader sequence (hereinafter, also referred to as "RAmylA leader sequence") is preferred inter alia.

[12] As to the gene coding for the hCTLA4-Ig fusion protein in accordance with the present invention, Ig of the hCTLA4-Ig is not particularly limited. Preferably, mention may be made of those consisting of hinge, CH and CH regions in Cγl of human IgGl, or those in which Cys, Cys and Cys in the hinge region are substituted 272 272 with Ser, respectively, or those in which Pro in the CH region is substituted with Ser.

[13] Also disclosed herein are recombinant vectors pMYN409, pMYN413 and pMYN414, comprising the hCTLA4-Ig fusion protein-coding genes, as shown in SEQ ID NO: 1, 2 and 3, respectively.

[14] Further disclosed herein are host cells transformed with the above-mentioned recombinant vectors pMYN409, pMYN413, and pMYN414, respectively. Preferably, the host cells are selected from the group consisting of rice (Oryza sativa L.), tobacco ( Nicotiana tabacum), maize (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), rape (Brassica napus) and potato ( Solanum tuberosum). Among these, more preferred is Oryza sativa L. Each Oryza sativa L. transformed with the recombinant vectors pMYN409, pMYN413 and pMYN414, respectively, was deposited with the Korean Collection for Type Cultures (KCTC) affiliated with the Korean Research Institute of Bioscience and Biotechnology (KRIBB, Korea), under Accession Numbers KCTC 10618BP (deposited on May 6, 2004), KCTC 10767BP (deposited on January 19, 2005) and KCTC 10768BP (deposited on January 19, 2005), respectively.

[15] Still further, disclosed herein is a process for producing a recombinant hCTLA4-Ig fusion protein, comprising suspension culturing the above-mentioned transformed host cells. Preferably, the suspension culture employs a basal medium, such as Chu N6, AA medium or MS (Murashige and Skoog) medium, containing 10 to 60 g/L of sucrose as a carbon source, 0.1 to 2.0 mg/L of 2,4-D and 0.01 to 2.0 mg/L of kinetin as growth regulators, and hygromycin for selection.

[16] Finally, disclosed herein is an hCTLA4-Ig fusion protein produced by the above- mentioned method.

[17] Hereinafter, the present disclosure will be further explained.

[18] Firstly, the present disclosure provides recombinant vectors pMYN409, pMYN413 and pMYN414 comprising the hCTLA4-Ig (cytotoxic T lymphocyte antigen 4-Immunoglobulin) fusion protein-coding gene. The recombinant vectors pMYN409, pMYN413, and pMYN414 comprise the RAmylA leader sequence and recombinant hCTLA4-Ig fusion protein-coding gene sequence, shown in SEQ ID NOs: 1, 2 and 3, respectively.

[19] In addition, recombinant vectors pMYN409, pMYN413, and pMYN414 are characterized by use of resistance to the antibiotic hygromycin as a selectable marker for transformed plant cells, and capability to secret recombinant hCTLA4-Ig fusion protein outside plant cells by the RAmylA leader sequence, upon suspension culturing (see Figs. 1, 8 and 9).

[20] Further, the Ig part present in the fusion protein hCTLA4-Ig comprises the hinge, CH and CH domains in the Cγl region of human IgGl.

[21] In the recombinant vector pMYN409 comprising the recombinant protein-coding gene shown in SEQ ID NO: 1 in accordance with the present invention, bases 1 to 93 in SEQ ID NO: 1 correspond to a signal peptide, bases 94 to 465 correspond to a CTLA4 extracellular region and bases 466 to 1166 correspond to an IgGl Fc fragment.

[22] Further, the present disclosure provides recombinant vectors pMYN413 and pMYN414, comprising SEQ ID NOs: 2 and 3, respectively, in which a portion of hCTLA4-Ig fusion protein-coding gene was altered from SEQ ID NO: 1. Specifically, SEQ ID NO: 2 is a sequence in which three amino acid codons of a hinge sequence of IgGl Fc fragment in the hCTLA4-Ig fusion protein-coding gene were altered by substitution of Cys with Ser. Whereas, SEQ ID NO: 3 is a sequence in which an amino 272 272 acid codon of a CH sequence of IgGl Fc fragment was substituted from Pro to Ser.

[23] Further, the present disclosure provides plant cells transformed with recombinant vectors pMYN409, pMYN413 and pMYN414 comprising the hCTLA4-Ig fusion protein-coding gene, respectively. Rice (Oryza sativa L. cv. Dongjin) is preferably utilized as the plant cell. As examples of other utilizable plant cells, mention may be made of tobacco (Nicotiana tabacum), maize (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), rape (Brassica napus) and potato (Solanum tuberosum).

[24] In accordance with the present disclosure, recombinant vectors pMYN409, pMYN413, and pMYN414 are transduced into the above-mentioned plant cells by a microprojectile bombardment-mediated transformation method and then only the transformed plant cell lines are selected from a selection medium.

[25] In addition, the present disclosure embraces a mass production method of prhCTLA4-Ig (plant recombinant human CTLA4-Ig), comprising suspension culturing the thus-selected transformed plant cells. That is, the antibiotic -resistant plant cells, transformed in the selection medium containing the antibiotic substance, were selected and suspension cultured. Preferably, suspension culture is carried out utilizing, as a basal medium, a Chu N6 or AA medium, or an MS (Murashige and Skoog) medium widely used in plant cell cultivation, containing 10 to 60 g/L of sucrose as a carbon source, 0.1 to 2.0 mg/L of 2,4-D and 0.01 to 2.0 mg/L of kinetin as growth regulators, and 50 mg/L of hygromycin as a selectable marker, at a temperature of 22 to 28°C and stirring at 80 to 150 rpm under dark conditions.

[26] As a result, the transformed plant cell lines express and secrete the prhCTLA4-Ig into the culture medium, upon suspension culturing. For example, when it is desired to culture Oryza sativa pMYN409 (KCTC 10618BP), this cell line is incubated at 28°C, under stirring at 120 rpm under dark conditions, using the N6 liquid medium or A A rice suspension culture medium, containing 30 g/L of sucrose, 2 mg/L of 2,4-D, 0.2 mg/L of kinetin and 50 mg/L of hygromycin. On day 7 of incubation, the culture medium is substituted with a sucrose-free N6 medium to induce expression of the recombinant protein, resulting in expression of prhCTLA4-Ig. When the Oryza sativa pMYN409 culture fluid is centrifuged to separate cell culture supernatant and purified by protein A affinity chromatography, followed by freeze-drying to obtain the prhCTLA4-Ig protein, the Oryza sativa pMYN409 (KCTC 10618BP) in accordance with the present invention yields about 10 mg of prhCTLA4-Ig per liter of culture fluid.

[27] Immunosuppressive activity of the prhCTLA4-Ig protein produced in accordance with the present invention was confirmed by T cell proliferation inhibitory effects through mixed Lymphocyte Reaction (MLR), ConA-induced T cell proliferation, and In Vitro T cell-dependent Antibody production experiments. Results thus obtained show that antiproliferative effects of the prhCTLA4-Ig protein on T cells were equal to or greater than those of recombinant hCTLA4-Ig protein produced via a conventional CHO cell expression system.

[28] Therefore, the present disclosure provides preparation of recombinant vectors comprising the hCTLA4-Ig fusion protein-coding gene and transformed plant cell lines utilizing the same, and induces prhCTLA4-Ig expression system of the transformed plant cell lines and thereby provides a mass production process of prhCTLA4-Ig having immunosuppressive activity. Consequently, the prhCTLA4-Ig can be produced on a large scale at lower production costs using the plant expression system and can also be utilized in medical applications due to confirmed immunosuppressive activity thereof.

[29] Brief Description of the Drawings

[30] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[31] Fig. 1 is a gene cleavage map of a pMYN409 vector in accordance with the present invention;

[32] Fig. 2A is a photograph showing PCR amplification results of hCTLA4-Ig genes contained in eleven transformed strains, and Fig. 2B is a photograph showing Southern blotting results of the amplified PCR products;

[33] Fig. 3 graphically shows quantification analysis results of prhCTLA4-Ig expressed in calli of the oryza sativa pMYN409 (KCTC 10618BP) prepared according to the present invention; [34] Fig. 4 graphically shows quantification analysis results of prhCTLA4-Ig expressed in suspension culture of the oryza sativa pMYN409 (KCTC 10618BP) prepared according to the present invention; [35] Fig. 5A is a photograph showing electrophoretic patterns of prhCTLA4-Ig protein in accordance with the present invention, and Fig. 5B is a photograph showing western blot patterns of prhCTLA4-Ig protein in accordance with the present invention; [36] Fig. 6 is a graph showing antiproliferative effect of prhCTLA4-Ig protein in accordance with the present invention on in- vitro T cells; [37] Fig. 7 is a graph showing antibody production inhibitory effects of prhCTLA4-Ig protein in accordance with the present invention on in-vitro T cell-dependent antibody production; [38] Fig. 8 is a gene cleavage map of a pMYN413 vector in accordance with the present invention; and [39] Fig. 9 is a gene cleavage map of a pMYN414ion on ConA-induced T cells;

[40] Fig. 10 shows SEQ ID NO : 1 in accordance with the present invention.

[41] Fig. 11 shows SEQ ID NO : 2 in accordance with the present invention.

[42] Fig. 12 shows SEQ ID NO : 3 in accordance with the present invention.

[43] Fig. 13 shows SEQ ID NO : 4, the amino acid sequence of hCTLA4-Ig fusion protein obtainable from the pMYN409 vector in accordance with the present invention. [44] Fig. 14 shows SEQ ID NO : 5, the amino acid sequence of hCTLA4-Ig fusion protein obtainable from the pMYN413 vector in accordance with the present invention. [45] Fig. 15 shows SEQ ID NO : 6, the amino acid sequence of hCTLA4-Ig fusion protein obtainable from the pMYN414 vector in accordance with the present invention. [46] Best Mode for Carrying Out the Invention [47]

[48] EXAMPLES

[49] Now, the present disclosure will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention. [50]

[51] Example 1 : Construction of vectors PMYN409. PMYN413 and PMYN414

[52] [53] Gene cleavage maps of recombinant expression vectors pMYN409, pMYN413 and pMYN414 expressing hCTLA4-Ig are shown in Figs. 1, 8 and 9, respectively.

[54] Firstly, to cause the hCTLA4-Ig fusion protein to be secreted into the culture medium, a RAmylA leader sequence was ligated to the N-terminus of hCTLA4 using overlapping polymerase chain reaction, thereby constructing a synthetic fusion gene.

[55] Utilizing a forward primer CTLA4-F1 (5'-TCCAACTTGACAGCCGGGGCAA TGCACGTGGCCCAGCCTGC-3'), concomitantly containing a C-terminal gene sequence of RAmylA signal sequence and an N-terminal gene sequence of CTLA-4, and a reverse primer CTLA4-R1 (5'-CTCTGCAGAATCTGGG CACGGTTCTG-3') recognizing the C-terminal region of CTLA-4, PCR was carried out as follows: one cycle of pre-denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds and elongation at 72°C for 45 seconds; and one cycle of final elongation at 72°C for 5 min.

[56] Using the thus-obtained PCR products, and a RAmylA secretion signal gene obtained from rice cDNA as a template, overlapping PCR was carried out to prepare a synthetic fusion gene in which a high secretion leader sequence of the rice amylase was bound to the hCTLA4 gene and the thus-prepared fusion gene was inserted into a pGEM-T Easy vector (Promega, USA) to construct a vector pMYN407.

[57] A pMYN406 vector including a human immunoglobulin IgGl Fc gene was cleaved with a restriction enzyme PstllSall and the resulting IgGl Fc gene was inserted into the pMYN407 vector which had been cleaved with the same restriction enzyme, thereby constructing a vector pMYN408.

[58] Finally, an expression vector pMYN409 was constructed by cleaving an hCTLA4-Ig fusion protein gene linked to the RAmylA secretion signal on the pMYN408 vector with BamHIISacI, and inserting the BamHIISacI fragment into a pMYN75 expression vector containing the same restriction enzyme cleavage sites to construct a pMYN409 vector. Insertion of the hCTLA4-Ig gene into the pMYN409 vector between a RAmy3D promoter and 3'-UTR region was confirmed through restriction enzyme mapping.

[59] Another expression vector, pMYN413, was also constructed as follows. Utilizing a Transformer Site-Directed Mutagenesis kit (BD Biosciences, USA), a vector pMYN410 containing a gene in which amino acids Cys, Cys and Cys in the hinge region of IgGl Fc had been substituted with Ser, respectively, was constructed from the pMYN408 vector, and then the resulting vector pMYN410 was cleaved with Nsil. The resulting fragment containing the substituted gene was inserted into the expression vector pMYN409 which had been cleaved with the same restriction enzyme, thereby constructing the vector pMYN413.

[60] Similarly, an expression vector pMYN414 was constructed as follows. Using the Transformer Site-Directed Mutagenesis kit(BD Biosciences, USA), a vector 272 pMYN411 containing a gene in which amino acid Pro in the CH region of IgGl Fc 272 was substituted with Ser, was constructed from the pMYN408 vector, and cleaved with Sad. The resulting fragment containing the substituted gene was inserted into the expression vector pMYN409 that had been cleaved with the same restriction enzyme, thereby constructing the vector pMYN414.

[61]

[62] Example 2 : Preparation of transformed plant cell line

[63]

[64] In order to introduce an hCTLA4-Ig gene into rice (Oryza sativa L. cv. Dongjin), transformation utilizing microprojectile bombardment was carried out with expression vectors pMYN409, pMYN413 and pMYN414.

[65] Firstly, rice seeds were dehulled, surface- sterilized and washed with sterile distilled water. The thus-treated rice seeds were placed on a callus-induction agar medium. After incubation at 25°C under a 16/8-hr light/dark cycle for 7 to 10 days, scutellum- derived calli were taken from germinated seeds and transferred to a fresh agar medium, followed by bombardment using a Biolistic PDS-1000/He (BioRad Laboratories, USA). After bombardment, the bombarded calli were further incubated in the dark at 25°C for 4 days, and were transferred and incubated in a selection medium (N6 SE) to which 50 mg/L of hygromycin B had been added. Transformed rice callus cell lines were obtained by incubating for 3 to 5 weeks until the hygromycin-resistant calli grew to a diameter of about 1 cm.

[66]

[67] Experimental Example 1 : Confirmation of gene introduction by Southern blot analysis

[68]

[69] In order to confirm whether insertion of the hCTLA4-Ig gene was successful in eleven transformed rice calli through the above-mentioned bombardment process, southern blotting was carried out in conjunction with genomic DNA PCR.

[70] Firstly, genomic DNA was isolated from rice calli using a DNAeasyplant mini kit (Qiagen, CA, USA). PCR amplification was carried out using the isolated genomic DNA as a template. Forward and reverse primers employed were CTLA4-F1 (5'-TCCAACTTGACAGCCGGGGCAATGCACGTGGCCCAGCCTGC-3') and hlgG-Rl (5'-CTCTAGACTCATTTACCCGGAGACAGGGAG-3'), respectively. The resulting PCR products were stained with EtBr after electrophoresis on a 1.0% agarose gel.

[71] Fig. 2 A is a photograph of PCR amplified hCTLA4-Ig gene contained in eleven transformed rice callus cell lines. M : a size marker, P : PCR positive control of pMYN409 vector, N : PCR negative control of genomic DNA of non-transformed rice cell, and Lanes 1 through 11 : PCR results of transformed rice callus cell lines of Example 2. The PCR amplified DNA segment exhibited a size of about 1167 bp, thereby confirming consistency with the expected size. [72] In addition, the PCR products were subjected to electrophoresis and then transferred to a Hybond -N+nylon membrane (Amersham Pharmacia, USA) for Southern blotting analysis. Hybridization was carried out using a- 32 P-labeled hCTLA4 DNA as the probe for Southern blotting. As can be seen from Fig. 2B, it was confirmed that the hCTLA4 DNA probe was combined with the PCR products amplified from the transformed cell lines. Therefore, it was determined that hCTLA4-Ig genes were successfully inserted into the chromosomes of all eleven transformed rice callus cell lines of Example 2, and as a result, among transformed calli, the transformed callus to which the gene of interest was normally introduced and in which gene expression rate was highest was designated as Oryza sativa pMYN409. Oryza sativa pMYN409 expressing prhCTLA4-Ig in accordance with the present invention was deposited with the Korean Collection for Type Cultures (KCTC) affiliated with the Korean Research Institute of Bioscience and Biotechnology (KRIBB, Korea) under Accession No. KCTC 10618BP (deposited on May 6, 2004).

[73]

[74] Experimental Example 2 : Quantification Analysis of transformed rice callus- derived recombinant hCTLA4-Ig fusion protein

[75]

[76] In order to quantify expression of hCTLA4-Ig protein of all eleven transformed rice callus-cell lines obtained in Example 2 and in order to select high-expression cell lines, ELISA (enzyme linked immunosorbent assay) was carried out.

[77] Firstly, rice calli were transferred to an N6 agar medium containing no sucrose so as to induce protein expression via the RAmy3D promoter. After crushing rice calli maintained on the N6 agar medium for 7 days, the resulting material was suspended in 500 D of phosphate buffered saline(PBS), vortexed for 1 minute and centrifuged at 15,000 rpm, 4°C for 30 minutes to separate a supernant which was then used for analysis.

[78] For ELISA, goat anti-human IgG (available from KPL) was diluted to 1:1000 in a coating buffer and 100 D/well was aliquoted into a 96-well plate. After overnight incubation at 4°C, wells were washed three times with a washing buffer, PBST (0.05% tween 20-containing PBS). 200 D of assay diluent (PBS buffer containing 2% FBS) was aliquoted into each well of the 96-well plate, reacted at room temperature for 1 hour and then washed with the washing buffer PBST three times. 100 D/well of callus extract samples was aliquoted, 2-fold serially diluted with assay diluent and reacted at 37°C for 1 hour. The reacted callus extracts were washed again with the washing buffer three times, and peroxidase-labeled goat anti-human IgG (available from KPL) diluted to 1:1000 in the assay diluent was added to 100 D/well, followed by reaction at 37°C for 1 hour. This was followed by washing with the washing buffer three times, reacting for 15 min with 100 D of a substrate and measuring absorbance at 405nm.

[79] Fig. 3 graphically represents ELISA quantification analysis results on recombinant hCTLA4-Ig fusion proteins expressed in transformed rice callus cell lines. BR-N002 cell line, which exhibited the highest expression among the eleven cell lines, produced about 54 D of recombinant hCTLA4-Ig per gram of fresh calli. The expression amount of hCTLA4-Ig per the total proteins was shown to be about 30 D of CTLA4-Ig/mg of total protein, thus confirming a protein expression level of about 3%.

[80]

[81] Example 3 : Suspension Culture Induction and Production of hCTLA4-Ig in medium

[82]

[83] Transformed rice callus-cell lines obtained in Example 2 were suspended in liquid culture media. 2 week- interval subculturing was carried out using an N6 liquid medium or AA rice suspension culture media, containing 30 g/L of sucrose, 2 mg/L of 2,4-D, 0.2 mg/L of kinetin, 50 mg/L of hygromycin, with incubating at 28°C, 120 rpm under dark conditions.

[84] In order to confirm secretion of the hCTLA4-Ig protein into the medium, protein expression was induced by exchange of the culture medium with a sucrose-free medium after 10-day s of incubation. 2 or 3 days after medium replacement, the culture medium was harvested and subjected to ELISA so as to confirm the amount of the hCTLA4-Ig protein secreted into the medium.

[85] Fig. 4 graphically shows cell growth and production of the hCTLA4-Ig fusion protein in the medium, when transformed rice suspension cell lines were cultured in the N6 medium (open square) and the AA medium (closed circle). When the culture media were replaced with sucrose free media to induce expression of recombinant proteins on day 7 of culture, production of the hCTLA4-Ig in the media rapidly increased 2 or 3 days later and it was confirmed that the hCTLA4-Ig fusion protein was produced up to about 10 mg/L.

[86]

[87] Experimental Example 3 : Assay of hCTLA4-Ig fusion protein

[88]

[89] 1. SDS-PAGE Electrophoresis and Western Blotting

[90]

[91] After suspension culture of Oryza sativa pMYN409, hCTLA4-Ig protein was recovered from the culture medium and purified using a protein A column. Purity and molecular weight of the purified prhCTLA4-Ig were determined by 10% denaturing SDS-PAGE electrophoresis and western blotting.

[92] Fig. 5A shows the electrophoretic patterns of prhCTLA4-Ig in SDS-PAGE, wherein lane 1 is a molecular weight marker and lane 2 represents prhCTLA4-Ig. As can be seen from Fig. 5A, the molecular weight of prhCTLA4-Ig was confirmed to be about 50kDa. Fig. 5B shows western blot patterns of the prhCTLA4-Ig. Herein, the prhCTLA4-Ig was transferred to a PVDF membrane after electrophoresis, and was reacted with goat anti-human IgG antibody (available from KPL, USA) as a primary antibody. The membrane was washed, and reacted with alkaline phosphatase- conjugated rabbit anti-goat IgG antibody (available from KPL, USA), followed by membrane washing. The washed membrane was appropriately reacted with a substrate and washed with tap water to stop the reaction. As can be seen from the photograph shown in Fig. 5B, reaction with the specific antibody confirmed that the prhCTLA4-Ig fusion protein has a molecular weight of about 50kDa.

[93]

[94] 2. Kinetic Analysis of hCTLA4 B7

[95]

[96] Interaction and affinity between plant cell-derived hCTLA4-Ig and B7-1 or B7-2 were confirmed using a BIAcore 3000(available from Biacore AB). As a chip for immobilizing a ligand, a CM5 sensor chip was employed. As the ligand for immobilizing on the sensor chip, B7-1 and B7-2 were employed. As the Analytes, CD28 (available from R & D system, controls), CHO cell-expressed hCTLA4-Ig and plant cell-derived hCTLA4-Ig were employed, respectively. Immobilization of ligand on the sensor chip was carried out by injecting the ligand diluted in 10 mM sodium acetate buffer, pH 5.5. Herein, PBS was employed as a running buffer and flow rate was maintained at 5 D/ml.

[97] The two analytes were reacted with ligands at 25°C and a flow rate of 30 D/ml for 2 minutes, respectively. After completion of analyte injection, bound ligands and analytes were artificially separated using 1M NaCl/50 mM NaOH.

[98]

[99] Table 1

[100] Summary of apparent kinetic constants

[101]

[102] As can be seen from table 1, association capability between respective ligands and analytes exhibited similar behavior. For ligand B7-1, A and P exhibited an approximately 10-fold higher association rate than CD28. Whereas, for ligand B7-2, A and P exhibited approximately 100-fold and 150-fold higher association rate than CD28, respectively. In addition, the dissociation rate was also higher in A and P, as compared to CD28. Both A and P exhibited affinity for ligand B7-1 that is about 2-fold higher than CD28, respectively. Whereas, for ligand B7-2, A and P exhibited approximately 6-fold and 8-fold higher affinity than CD28, respectively. As a result, it was confirmed that both CHO-expressed hCTLA4-Ig and prhCTLA4-Ig had higher affinity for the B7 family (B7-1 and B7-2) than CD28, and prhCTLA4-Ig had an affinity for the B7 family (B7-1 and B7-2) similar to that of CHO-expressed hCTLA4-Ig.

[103] [104] 3. Inhibition of ConA-induced T cell proliferation [105] [106] In order to determine T cell proliferative ability, thespleen was excised from aBDFl mouse and splenocytes were harvested therefrom. Cell concentration was adjusted to 1 X 10 cells/ml and 200 D/well was aliquoted and incubated on a 96-well plate. CHO cell-derived hCTLA4-Ig and prhCTLA4-Ig were added to 0.001, 0.01, 0.1, 1 and 10 D/ml concentrations thereto. This was followed by addition of 5 D/ml of ConA, a mitogen that induces proliferation of T lymphocytes, and incubation in a 5% CO incubator at 37°C for 3 days. Prior to completion of incubation, [ H]-thymidine was added and cells were harvested from each well using an Automatic Cell Harvester. Then, the degree of immunocyte proliferation was examined by measuring the degree of incorporation of [ H]-thymidine into DNA. [107] As can be seen from Fig. 6, in comparison with a media control (VH), both CHO cell-derived hCTLA4-Ig and prhCTLA4-Ig inhibited ConA-induced T cell proliferation in a concentration-dependent manner, and exhibited antiproliferative effects of more than 50% at concentrations greater than 1 D/ml. In addition, the inhibition degree of prhCTLA4-Ig on T cell proliferation was almost the same as that of CHO cell-derived hCTLA4-Ig.

[108]

[109] 4. Inhibition of in vitro T cell-dependent antibody production

[HO]

[111] Activation of splenocytes by use of T cell-dependent antigens, sheep red blood cells (sRBCs), leads to generation of plasma cells producing antibodies against sRBCs. Splenocytes were adjusted to a concentration of 1 X 10 cells/ml and 0.5 ml/well was aliquoted to a 48-well plate. Thereafter, sRBCs were diluted to 1 X 10 cells/ml using a buffer (EBSS) and 6.5 D aliquots of sRBC were added to each well. This was followed by incubation for 5 days and recovery of cultured cells for use in plaque assay.

[112] 0.85% agar was treated with plasma cells producing antibodies against sRBCs, sRBCs as target cells, guinea pig complements and CHO cell-derived hCTLA4-Ig and prhCTLA4-Ig in concentrations of 0.001, 0.01, 0.1, 1 and 10 D/ml, respectively, and incubated in a 5% CO incubator at 37°C for about 1 hour. The plaques were counted and calculation was made in terms of PFC/10 cells. The more activated plasma cells, the higher the antibody production. Action of guinea pig complements lead to lysis of sRBCs around cells, thereby resulting in increased plaques.

[113] As can be seen from Fig. 7, upon comparing with a media control (VH), both CHO cell-derived CTLA4-Ig and prhCTLA4-Ig inhibited antibody production of plasma cells against sRBCs which are T cell-dependent antigens, in a concentration-dependent manner, and exhibited inhibitory effects of more than 80% at concentrations greater than 1 D/ml. In addition, the inhibition degree of prhCTLA4-Ig on antibody production of plasma cells against sRBCs which are T cell-dependent antigens, was almost the same as that of CHO cell-derived CTLA4-Ig.

[114] Industrial Applicability

[115] The present invention has established a process for producing a recombinant hCTLA4-Ig fusion protein having immunosuppressive activity, using transformed plant cell culture. Therefore, in accordance with the process of the present invention, it is possible to economically achieve mass production of the recombinant hCTLA4-Ig fusion protein having immunosuppressive activity.

[116] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modi- fications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Sequence Listing

[117] BRP05642.app

[118]

[119]

Till: pϋivro IWPIiNATlONAL. UORU RECEIPT IN THE CASE OF AN ORIGWAX, DEPOSIT issued pursuant to Rule 7,1 TO Rnryuπβ Phar Ui., LTD BoryuriR Hlda, #βo-21, WBII* dang, Chonaro-ku, Seoul ]]fh?S0, Republic of orea

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roa mi i uiuOsr or mτPNT i KOetoi in INIEΠNATIONAI IΌHM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7 I 10 Boryuns Hnrrn Co LID Borv nβ Blφf Sfi6-2l Wonnam dona Chongro-gu, Seoul 110-750 Republic of Korea

raπ rM iKhAiY o.\ ot_' 'i iiF Or iUICR 0Tϊt.AMΪ>MS I'UR lllp Pi rXK OF PATENT PHOCKU Ullh INTERNATIONAL 1'OKM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7 1

TO ^■ Boiyung Phaim Co. LTD Boryung Bldg, jffiG-21 Woπtiam-dorig, ChongiO-εu, Seoul 110 750. Republic of Korea I . IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR^' INTERNATIONAL DEPOSITARY AUTHORITY Oryza saliva /pMYW414 (rice callus cell line) KCTC 10768RP n sci MTinc Dϋbemrno.N AND/OK PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was. accompanied by [ x ] a scienlilic description T ] a proposed taxonomic designation (Mark with a cross where applicable) ffl RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under I abov w whhiirchh w w;aιss r rpercpeiivvpeHd h bvy i itt n onn J Iaann 1199 2200005-. 1Y RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depository Authority on and a request to convert the original (lepos.it to a deposit under the Budapest Treaty was received by it on V IN KUNATIONAI. DEPOSITARY AUTHORITY Name- Korean Collection for Type Cultures

Address Korea Kesearch Institute of Bioscience and Biotechnology (KRIBB) *62, Oun-duπR, YuS.ong-ku, Taejon 305-333, Republic of Korea

ronn BP'4 IKC Te rrain 17)

Claims

[I] A gene coding for a human cytotoxic T lymphocyte antigen 4-Immunoglobulin (hCTLA4-Ig) fusion protein comprising at least one plant-derived leader sequence selected from the group consisting of rice (Oryza sativa L.), tobacco ( Nicotiana tabacum), maize (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), rape (Brassica napus) and potato ( Solanum tuberosum).

[2] The gene according to claim 1, wherein the plant-derived leader sequence is a RAmylA leader sequence.

[3] The gene according to claim 1, wherein, in the hCTLA4-Ig, Ig is hinge, CH ,and CH regions of Cγl of human IgGl.

[4] The gene according to claim 3, comprising the sequence shown in SEQ ID NO: 1.

[5] A recombinant vector pMYN409 comprising the hCTLA4-Ig fusion protein- coding gene as claimed in claim 4.

[6] A host cell transformed with the recombinant vector pMYN409 as claimed in claim 5.

[7] The host cell according to claim 6, wherein the host cell is selected from the group consisting of rice (Oryza sativa L.), tobacco (Nicotiana tabacum), maize ( Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato ( Lycopersicon esculentum), rape (Brassica napus) and potato (Solanum tuberosum).

[8] The host cell according to claim 7, wherein the host cell is Oryza sativa L. (Accession No. KCTC 10618BP).

[9] A process for producing a recombinant hCTLA4-Ig fusion protein, comprising suspension culturing the host cell as claimed in claim 8.

[10] The process according to claim 9, wherein the suspension culturing employs a basal medium, such as Chu N6, AA or MS (Murashige and Skoog) medium, containing 10 to 60 g/L of sucrose as a carbon source, 0.1 to 2.0 mg/L of 2,4-D and 0.01 to 2.0 mg/L of kinetin as growth regulators, and hygromycin for selection.

[I I] An hCTLA4-Ig fusion protein produced by the process as claimed in claim 9.

[12] The gene according to claim 3, wherein Cys, Cys and Cys in the hinge are substituted with Ser, respectively.

[13] The gene according to claim 12, comprising the sequence shown in SEQ ID NO: 2.

[14] A recombinant vector pMYN413 comprising the hCTLA4-Ig fusion protein- coding gene as claimed in claim 13.

[15] A host cell transformed with the recombinant vector pMYN413 as claimed in claim 14.

[16] The host cell according to claim 15, wherein the host cell is selected from the group consisting of rice (Oryza sativa L.), tobacco (Nicotiana tabacum), maize ( Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato ( Lycopersicon esculentum), rape (Brassica napus) and potato (Solanum tuberosum).

[17] The host cell according to claim 16, wherein the host cell is Oryza sativa L. (Accession No. KCTC 10767BP).

[18] A process for producing a recombinant hCTLA4-Ig fusion protein, comprising suspension culturing the host cell as claimed in claim 17.

[19] The process according to claim 18, wherein the suspension culturing employs a basal medium, such as Chu N6, AA or MS (Murashige and Skoog) medium, containing 10 to 60 g/L of sucrose as a carbon source, 0.1 to 2.0 mg/L of 2,4-D and 0.01 to 2.0 mg/L of kinetin as growth regulators, and hygromycin for selection.

[20] An hCTLA4-Ig fusion protein produced by the process as claimed in claim 18. 272

[21] The gene according to claim 3, wherein Pro in the CH domain is substituted with Ser.

[22] The gene according to claim 21, comprising the sequence shown in SEQ ID NO: 3.

[23] A recombinant vector pMYN414 comprising the hCTLA4-Ig fusion protein- coding gene as claimed in claim 22.

[24] A host cell transformed with the recombinant vector pMYN414 as claimed in claim 23.

[25] The host cell according to claim 24, wherein the host cell is selected from the group consisting of rice (Oryza sativa L.), tobacco (Nicotiana tabacum), maize ( Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato ( Lycopersicon esculentum), rape (Brassica napus) and potato (Solanum tuberosum).

[26] The host cell according to claim 25, wherein the host cell is Oryza sativa L. (Accession No. KCTC 10768BP).

[27] A process for producing a recombinant hCTLA4-Ig fusion protein, comprising suspension culturing the host cell as claimed in claim 26.

[28] The process according to claim 27, wherein the suspension culturing employs a basal medium, such as Chu N6, AA or MS (Murashige and Skoog) medium, containing 10 to 60 g/L of sucrose as a carbon source, 0.1 to 2.0 mg/L of 2,4-D and 0.01 to 2.0 mg/L of kinetin as growth regulators, and hygromycin for selection.

[29] An hCTLA4-Ig fusion protein produced by the process as claimed in claim 27.