KR101193940B1 - Rhesus monkey CTLA-4 fusion Ig - Google Patents

Abstract

The present invention relates to a rhesus monkey CTLA-4 fusion immunoglobulin, and more specifically, to a rhesus monkey CTLA-4 extracellular domain having an amino acid sequence of the 38th to 161st amino acids of SEQ ID NO: 2, and A rhesus monkey CTLA-4 fused immunoglobulin consisting of the Fc region of human immunoglobulin (human IgG).

According to the present invention, the rhesus monkey CTLA-4 fused immunoglobulin may be prepared, and when the rhesus monkey CTLA-4 fused immunoglobulin is treated to T cells of rhesus monkey, It was found to inhibit T cell activity more effectively than CTLA-4 fusion protein. Therefore, by using the rhesus monkey CTLA-4 fused immunoglobulin of the present invention, it is possible to increase the efficiency of immunosuppression in the allogeneic transplantation experiment using the rhesus monkey, and to proliferate immune cells comprising the fusion immunoglobulin as an active ingredient. The use of inhibitors is expected.

Description

Rhesus monkey CTLA-4 fusion immunoglobulin {Rhesus monkey CTLA-4 fusion Ig}

The present invention relates to a rhesus monkey CTLA-4 fusion immunoglobulin, and more specifically, to a rhesus monkey CTLA-4 extracellular domain having an amino acid sequence of the 38th to 161st amino acids of SEQ ID NO: 2, and A rhesus monkey CTLA-4 fused immunoglobulin consisting of the Fc region of human immunoglobulin (human IgG).

The activity of T lymphocytes in the immune system plays an important role in regulating several different immune system cells. In order for these T lymphocytes to be active, two signals must be transmitted from antigen presenting cells (APCs). In other words, co-stimulatory signals are essential along with signals that antigen-presenting cells recognize antigens and present antigens to T cell antigen receptors (TCRs) through major histocompatibility complex (MHC). Only then can T cells become active. One such known costimulatory signal is CD28 expressed in T cells. CD28 is a 44 KD glycoprotein that binds to CD80 (B7-1) and CD86 (B7-2) expressed in APCs to produce cytokine, transcriptional activity, and Increase cell survival, etc. (Lenschow et al., 1996).

In particular, CTLA-4 (Cytotoxic T lymphocyte-associated antigen 4; CD152) was first discovered in 1987 as a member of an immunoglobulin (Ig) large family that competitively acts on CD28. The CTLA-4 is expressed on the cell surface of the activated T cells and binds to CD80 and CD86 to serve to transmit signals to the T cells. CTLA-4 has a higher affinity for ligands than CD28 (Linsley et al., 1994). Unlike CD28, CTLA-4 binds to a ligand and mediates T cell activity suppression signal [Waterhouse et al., 1996], and T cell activated by reducing the expression of IL-2 and IL-2 receptors in T cells. Inhibits the activity and proliferation of [Krummel et al., 1996].

Since co-stimulatory factors such as CTLA-4 are essential elements for regulating the immune response of T cells, many studies have been conducted to control the immune response by regulating it. In particular, the immunomodulation using CTLA-4 co-stimulating factor is applied to the technology of recombinant antibodies (immunoglobulin fusion protein). For example, since CTLA-4 has high binding ability with CD80 and CD86, water-soluble CTLA-4 is produced and used as a co-stimulatory signal blocker through CD28 of T cells [Lenschow et al., 1992; Kosuge et al., 2003; Abrams et al., 1999], CTLA-4 using human and mouse CTLA-4 genes were invented and the effect of munoglobulin [Gerstmayer et al., 1997]. However, immunoglobulin fusion protein (Ig) using CTLA-4 of Rhesus monkey has not been invented yet, and its effect is unknown.

Accordingly, the present inventors have made intensive research to overcome the problems of the prior art, and prepared a rhesus monkey CTLA-4 fusion immunoglobulin using CTLA-4 of Rhesus monkey. When munoglobulin is applied to rhesus monkey T cells, it was confirmed that the activity of rhesus monkey T cells can be effectively inhibited, and the present invention has been completed.

Accordingly, a main object of the present invention is to provide a rhesus monkey CTLA-4 fusion immunoglobulin having the advantage of effectively inhibiting the activity of rhesus monkey T cells.

Another object of the present invention to provide an immune cell proliferation inhibitor using the rhesus monkey CTLA-4 fusion immunoglobulin.

According to one aspect of the invention, the invention comprises a rhesus monkey CTLA-4 extracellular domain and an Fc region of human immunoglobulin (Fc region) of human IgG -4 provides fusion immunoglobulins.

In the rhesus monkey CTLA-4 fusion immunoglobulin of the present invention, the extracellular domain of the rhesus monkey CTLA-4 (Genbank Accession No. NM_001044739.1) is capable of binding any rhesus monkey CTLA-4 capable of binding to CD28. It may also be an extracellular domain of, preferably characterized in that having the 38 to 161 th amino acid sequence of SEQ ID NO: 2.

In addition, the Fc region of the human immunoglobulin may be any region other than a fragment antigen binding region in which human immunoglobulins bind to leukocytes or macrophages, and may be any region capable of binding to leukocytes or macrophages. The sequence can be obtained from NCAA's AAA02914.

In addition, a hinge sequence, which is a flexible region, may be included between the extracellular domain of the rhesus monkey CTLA-4 and the human immunoglobulin Fc region. In the present invention, the hinge sequence is connected to the Fc region of human immunoglobulin. It was used as it is.

In rhesus monkey CTLA-4 fusion immunoglobulin of the present invention, the rhesus monkey CTLA-4 fusion immunoglobulin is characterized by having an amino acid sequence of SEQ ID NO: 4. In addition, the rhesus monkey CTLA-4 fusion immunoglobulin may be encoded by a gene having a nucleotide sequence of SEQ ID NO: 3.

In addition, the fusion immunoglobulin is characterized in that it further comprises a leader peptide (leader peptide) at the N 'end of the rhesus monkey CTLA-4 extracellular domain.

The term “leader peptide” of the present invention refers to a sequence present at the N-terminus of a secretory protein precursor and also not present in naturally mature protein, and the leader peptide refers to a peptide cleaved from the protein precursor. In general, leader sequences are cleaved by proteases (or signal peptides) upon secretion into the cell. Although such leader peptides have certain common sequence characteristics across species, leader peptides that secrete to one species do not necessarily exert secretory functions in other species. In the prepro protein or the free protein, the leader peptide may be from a different protein or may be a leader peptide naturally present in the protein of interest, preferably derived from the secreted protein of the host used. Alternatively, the codon may be modified to have an optimal codon according to the codon usage frequency of the host. In addition, leader peptides that can be used for the purposes of the present invention may contain a portion of the N-terminal amino acid sequence of the naturally occurring mature protein derived therefrom. In the present invention, a leader peptide having an amino acid sequence of “METDTLLLWVLLLWVPGSTGD” was specifically used.

According to another aspect of the present invention, the present invention provides a rhesus monkey CTLA-4 extracellular domain gene and human immunoglobulin (human IgG) encoding the 38-161 amino acid sequence of SEQ ID NO: 2. It provides a recombinant expression vector comprising the Fc gene of.

In the recombinant expression vector of the present invention, the Rhesus monkey CTLA-4 extracellular domain gene is characterized by having the 112-482 base sequence of SEQ ID NO: 1.

The recombinant expression vector of the present invention may be any replicable expression vector capable of expressing the rhesus monkey CTLA-4 fusion immunoglobulin, but preferably has a RheCTLA-4IgG having a cleavage map of FIG. / pLNCX2.

According to another aspect of the present invention, the ball invention provides a host cell transformed with the recombinant expression vector.

The host cell of the present invention may be any animal cell capable of expressing and secreting the rhesus monkey CTLA-4 fusion immunoglobulin, preferably CHO (chinese hamster ovary) cells, COS-7 (Monkey, African green) cells, NIH / 3T3 (Mouse fibroblast) cells, BHK-21 (Baby Hamster kidney, Syrian golden) cells, Hela (Human Carcinoma) cells, Vero (Monkey kidney, African green) cells, and C127 (Mouse) cells It is preferable to use any one of the cells selected from the group consisting of, more preferably using CHO cells due to the high transformation rate. In the embodiment of the present invention was transformed using 293GPG cells of the Tetracycline off system and CHO cells which are mammalian-derived cell lines. When transformed through the 293GPG cells, the expression was more stably maintained and transformed into CHO cells via the 293GPG cells because they had the advantage of little toxicity to the target cells.

According to one aspect of the invention, the present invention provides an immune cell proliferation inhibitor comprising the rhesus monkey CTLA-4 fusion immunoglobulin as an active ingredient.

In the immune cell proliferation inhibitor of the present invention, the allogeneic response is blocked by the rhesus monkey CTLA-4 fusion immunoglobulin.

The term "allogeneic response" of the present invention refers to an immune response that occurs in organ transplantation between the same species. An important antigen that causes an immune response is a major histocompatibility antigen (MHC), which acts as an antigen due to its high expression in cells. Therefore, when there is no similarity of MHC in transplantation between the same species, rejection by immune response occurs due to recognition as antigen.

In an embodiment of the present invention, when the activity is measured by treating the rhesus monkey CTLA-4 fusion immunoglobulin and mouse-derived CTLA-4 fusion protein to T cells of rhesus monkey, respectively, Globulin was confirmed to more effectively inhibit the activity of T cells (Example 4). Therefore, the use of species-specific rhesus monkey CTLA-4 fusion immunoglobulin than other CTLA-4 fusion immunoglobulins used previously can increase the efficiency of immunosuppression in allograft experiments using rhesus monkeys.

The immune cell proliferation inhibitor of the present invention may be prepared by a method known in the pharmaceutical field for use as a therapeutic agent, and may be mixed with a pharmaceutically acceptable carrier, excipient, diluent, and the like to powder, granule, tablet, capsule Or in the form of an injection or the like. They can also be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) or orally.

The therapeutic agent of the invention is administered in a therapeutically effective amount. The term “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the patient's age, sex, weight, health condition and symptoms of the disease. It can be appropriately selected depending on the administration time, administration method, preferably 0.01 ~ 100 mg per day of adult can be administered.

According to another aspect of the invention, the invention provides a method for inhibiting immune cell proliferation comprising treating rhesus monkey CTLA-4 fused immunoglobulin to immune cells in vitro.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

< Example  One. RT - PCR  Of genes by method Cloning  >

Peripheral Blood Mononuclear Cells (PBMCs) of Rhesus monkey were added 200 μl of chloroform (Chigroform, Sigma) per 1 ml of Trizol (Invitrogen) CDNA was replicated by performing reverse electron chain polymerization (RT-PCR) with 2 μg of mRNA as a template. Using the cDNA as a template to prepare a primer as shown in Table 1 (A) according to the base sequence obtained from the gene database, rhesus monkey CTLA-4 gene was obtained through a chain polymerization reaction. In order to obtain only the nucleotide sequence including the extracellular domain of the obtained rhesus monkey CTLA-4 gene, primers were prepared as in Table 1 (B) below and subjected to chain polymerization again. At this time, the reverse transcriptase was used Superscript III (Invitrogen), the polymerase was used Tag polymerase (gene craft). As a result, the primers and PCR products of Table 2 were obtained. 100% agreement with NM_001044739. The size of the PCR product is 399 bp.

Table 1. Primers

Table 2. Primers

< Example  2. Preparation of Protein Expression Plasmids Transformed cell line  Production>

In order to express the protein from Rhesus monkey CTLA-4 gene, Rhesus monkey CTLA-4 extracellular domain gene was transferred to pLNCX2 (FIG. 2 (A), CLONTECH) which is a mammalian expression vector. Cloned.

First, a primer for synthesizing the cleavage site sequence of restriction enzyme Xho I was synthesized at the 5 'end, and a restriction enzyme cleavage site was made by the polymerase chain reaction. Restriction cleavage sites were made by chain reaction. The cDNA of the Fc region of human immunoglobulin (human IgG) was then fused to the 3 ′ end of the rhesus monkey CTLA-4 extracellular domain gene. The cDNA is a cDNA obtained through reverse transcription after extracting mRNA from human peripheral lymphocytes. Restriction enzyme sites used for fusion were restriction enzyme BamHI at the 5 'end and restriction enzyme NotI at the 3' end. The base sequence of the restriction enzyme cleavage site is as follows.

XhoⅠ: CCG CTC GAG

BamHⅠ: GGA TCC

NotⅠ: GCG GCC GCC ACC

Here, lysine, which is the last amino acid of the human immunoglobulin Fc region gene sequence, was replaced with cysteine. Substitution of the amino acid causes palmitoylation to immunoglobulin, thereby enhancing the stability of the micelle structure when delivered in liposome form during in vivo delivery [Missirlis et al., 2009]. As a result, the recombinant vector RheCTLA-4IgG / pLNCX2 (FIG. 2B) expressing the rhesus monkey CTLA-4 extracellular domain fusion immunoglobulin having the structure of FIG. 1 was completed.

Thereafter, 293GPG cells of the Tetracycline off system and CHO (Chinese Hamster Ovary) cells, which are mammalian-derived cell lines, were transformed. First, 293GPG cells were seeded in 6 well tissue culture plates to 1 × 10 ⁵ cells per well, and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when cells grew to cover 70-80% of the well surface, 1 to 2 μg of rhesus macaque CTLA-4 fusion immunoglobulin expression vector DNA was added in serum-free DMEM medium. μg of Lipofectamine plus (Invitrogen) was used to introduce 293GPG cells. Thereafter, 293GPG cells into which the expression vector was introduced were incubated for 24 hours, and the host cells (CHO cells) were incubated for 4 to 5 hours using a culture supernatant containing a virus (retrovirus). It was incubated for 24 hours with the medium containing%, this process was repeated three times. The CHO cells were treated with G418 antibiotic 1.5 mg / ml to obtain the transduced cells to obtain cells expressing the gene.

< Example  3. Protein Expression and Purification>

To confirm that rhesus monkey CTLA-4 fusion immunoglobulin protein is overexpressed in mammalian cells, total RNA was extracted from transformed CHO cells. RNA extraction method is the same as the method of Example 1. Using cDNA as a template, the presence or absence of rhesus monkey CTLA-4 ECD gene expression was confirmed by polymerase chain reaction. In addition, the transformed cells were identified through an ELISA kit (R & D system) to identify the proteins expressed and secreted in the culture supernatant.

After confirming the expression of rhesus monkey CTLA-4 fusion immunoglobulin protein by the above method, in order to purify the expressed protein from the culture supernatant obtained by culturing the identified transformed cells, Protein A Agarose kit (553-50- 00, KPL). First, pass the wash / binding buffer (0.1M sodium phosphate, 0.15M NaCl, pH 7.4) to the Protein A Agarose column, and then pass the culture supernatant containing rhesus monkey CTLA-4 fused immunoglobulin protein. Adsorbed to the column using the binding force of immunoglobulin (IgG). Adsorbed proteins were eluted again using Elution buffer (0.2M Glycine, pH 3 ± 1.85). The eluted samples were subjected to SDS-polyacrylamide gel electrophoresis and stained with coomassie blue to identify the rhesus monkey CTLA-4 fused immunoglobulin protein expressed. The protein purification results are shown in FIG. 3.

Figure 3 (A) is the result of confirming through RT-PCR in order to confirm whether the expression vector is properly expressed after introduction into the host cell CHO cells. Primers and product sizes used are shown in Table 3 below. Rhesus macaque CTLA-4 fusion immunoglobulin and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were introduced in each of the cells to which the rhesus macaque CTLA-4 fusion immunoglobulin gene was expressed as mRNA. It was confirmed using a primer that matches the sequence. The GAPDH is a gene whose expression level does not change well depending on the conditions of the cell, and was used as an intracellular standard in the investigation of the expression level of fusion immunoglobulin of the present invention.

Figure 3 (B) shows the results confirmed by SDS-PAGE after culturing the CHO cells into which the gene is introduced and purified rhesus monkey CTLA-4 fusion immunoglobulin protein purified from the culture supernatant. Calculation using only the number of amino acids is about 40.8 KDa, but the size of the measured protein is found to be expressed in a slightly larger size than the size calculated by protein modification through glycosylation (glycosylation) It was. The identified proteins were dialyzed in PBS to reach equilibrium, and then stored at −70 ° C. and used for immunosuppression experiments.

[Table 3. Size of primer and PCR product]

< Example  4. Rhesus  Measuring the Inhibitory Effect of Monkey T Cells>

In order to measure the inhibitory effect of rhesus monkey T cells, the inhibition of immune cell function of rhesus monkeys was confirmed using the mixed lymphocyte reaction (MLR) method. In the present invention, the MHC haplotype reacts with another effector cell and a target cell to cause an immune response. In other words, in the case of organ transplantation, a long-term rejection reaction occurs. When the immune response occurs, T cells proliferate, and radioisotopes ³ H contained in the culture medium accumulate in the T cells during the proliferation of T cells. After the reaction, immune activity can be compared by measuring ³ H accumulated in the cells. The process is described in detail below.

First, T lymphocyte 1 × 10 ⁵ cells isolated from PBMCs of leather monkeys were used as effectors. In addition, PBMC 1 × 10 ⁵ cells of other leather monkeys having different MHC haplotypes were irradiated to inactivate the cells, and then cultured with the effector cells as target cells. After 24, 48 and 72 hours of culture, the inhibitory effect of T cells was measured. At this time, ³ H marked radioisotopes were treated with effector cells 12 hours ago, and the amount of radioisotope taken up from the divided cells was measured. The measurement results are shown in FIG. 4.

As shown in Figure 4, it can be observed that leather monkey monkey CTLA-4 fusion immunoglobulin protein inhibits the activity of leather monkey monkey T cells more effectively than the mouse-derived CTLA-4 fusion immunoglobulin protein.

As described above, according to the present invention, antibody recombinant protein (fusion immunoglobulin) can be prepared using CTLA-4 of Rhesus monkey.

As seen through the embodiment of the present invention, when the rhesus monkey CTLA-4 fusion immunoglobulin of the present invention is treated to rhesus monkey T cells, T is more effective than other conventional CTLA-4 fusion proteins. It was confirmed to inhibit the activity of the cells. Therefore, by using the species-specific rhesus monkey CTLA-4 fusion immunoglobulin of the present invention, it is possible to increase the efficiency of immunosuppression during allograft experiment using rhesus monkey, and comprising the fusion immunoglobulin as an active ingredient. The use of immune cell proliferation inhibitors is expected.

1 shows a schematic diagram of gene construction for the expression of rhesus monkey CTLA-4 immunoglobulin.

2 (A) shows a cleavage map of the expression vector pLNCX2, and (B) shows a cleavage map of the recombinant vector RheCTLA-4IgG / pLNCX2 comprising DNA encoding rhesus monkey CTLA-4 fused immunoglobulin protein. .

3 shows the results of rhesus monkey CTLA-4 fusion immunoglobulin protein purification.

Figure 4 shows the results of inhibition of rhesus monkey T cells activity of rhesus monkey CTLA-4 fusion immunoglobulin protein.

<110> Korea University Industrial & Academic Collaboration Foundation <120> Rhesus monkey CTLA-4 fusion Ig <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 672 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for Rhesus monkey CTLA-4 <400> 1 atggcttgcc ttggatttca gcggcacaag gctcggctca acctggctac caggacccgg 60 ccctacactc tcctgttttc tcttctcttc atccctgtct tctccaaagc aatgcacgtg 120 gcccagcctg ctgtggtgct ggccaacagc cgagggatcg ccagctttgt gtgtgagtat 180 gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc cgacagccag 240 gtgactgaag tctgtgcggc aacatacatg atggggaatg agttgacctt cctagatgat 300 tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 360 gctatggaca caggactcta catctgcaag gtggagctca tgtacccacc accatactac 420 atgggcatag gcaatggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 480 gacttcctcc tctggatcct tgcagcagtt agttcggggt tgttttttta tagctttctc 540 ctcacagctg tttctttgag caaaatgcta aagaaaagaa gccctctcac aacaggggtc 600 tatgtgaaaa tgcccccaac agagccagaa tgtgaaaagc aatttcagcc ttattttatt 660 cccatcaatt ga 672 <210> 2 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for Rhesus monkey CTLA-4 <400> 2 Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Arg Leu Asn Leu Ala   1 5 10 15 Thr Arg Thr Arg Pro Tyr Thr Leu Leu Phe Ser Leu Leu Phe Ile Pro              20 25 30 Val Phe Ser Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala          35 40 45 Asn Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly      50 55 60 Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln  65 70 75 80 Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr                  85 90 95 Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val             100 105 110 Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile         115 120 125 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Met Gly Ile Gly     130 135 140 Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser 145 150 155 160 Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe                 165 170 175 Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys             180 185 190 Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu         195 200 205 Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn     210 215 220 <210> 3 <211> 1428 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for Rhesus monkey CTLA-4 fusion immunoglobulin <400> 3 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gactttactg tcacggttcc caaggaccta tatgtggtag agtatggcag caatatgaca 120 attgaatgca gattcccagt agaaaaacaa ttaggcctga cttcactaat tgtctattgg 180 gaaatggagg ataagaacat tattcaattt gtgcatggag aggaagacct gaaggttcag 240 catagtaact atagacagag ggcccagctg ttgaaggacc agctctccct gggaaatgct 300 gcacttcgga tcacagatgt gaaattgcag gatgcagggg tttaccgctg catgatcagc 360 tatggtggtg ccgactacaa gcggattacc gtgaaagtca atgctccata caacaaaatc 420 aaccaaagaa ttttggttgt cgatccagtc acctctgaac atgaactaac atgtcaggct 480 gagggctacc ccaaggccga agtcatttgg acaagcagtg accatcaagt cctgagtggt 540 aagaccacca ccaccaattc caagagagag gagaagcttt taaatgtgac cagcacactg 600 agaatcaaca caacagctaa tgagattttc tactgcattt ttaggagatt aggtcctgag 660 gaaaaccata cagctgaatt ggtcatccca gaactacctc tggcgcttcc tccaaatgaa 720 aggggatccg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1140 ccatcacgag atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1320 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gttgttga 1428 <210> 4 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> Amino acid for Rhesus monkey CTLA-4 fusion immunoglobulin <400> 4 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Asp Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val              20 25 30 Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Arg Phe Pro Val Glu          35 40 45 Lys Gln Leu Gly Leu Thr Ser Leu Ile Val Tyr Trp Glu Met Glu Asp      50 55 60 Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln  65 70 75 80 His Ser Asn Tyr Arg Gln Arg Ala Gln Leu Leu Lys Asp Gln Leu Ser                  85 90 95 Leu Gly Asn Ala Ala Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala             100 105 110 Gly Val Tyr Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg         115 120 125 Ile Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile     130 135 140 Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala 145 150 155 160 Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln                 165 170 175 Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys             180 185 190 Leu Leu Asn Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Ala Asn Glu         195 200 205 Ile Phe Tyr Cys Ile Phe Arg Arg Leu Gly Pro Glu Glu Asn His Thr     210 215 220 Ala Glu Leu Val Ile Pro Glu Leu Pro Leu Ala Leu Pro Pro Asn Glu 225 230 235 240 Arg Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro                 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro             260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr         275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn     290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val                 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser             340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys         355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp     370 375 380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu                 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe             420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly         435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr     450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Cys 465 470 475  

Claims (10)

Consisting of Rhesus monkey CTLA-4 extracellular domain having an amino acid sequence of amino acids 38-161 of SEQ ID NO: 2 and an Fc region of human immunoglobulin (human IgG); Rhesus monkey CTLA-4 fusion Ig, characterized by having the amino acid sequence of No. 4. delete Recombinant expression vector comprising a gene encoding rhesus monkey CTLA-4 fusion immunoglobulin according to claim 1. The recombinant expression vector according to claim 3, wherein the gene encoding Rhesus monkey CTLA-4 extracellular domain has the 112 th to 482 th nucleotide sequences of SEQ ID NO: 1. The recombinant expression vector of claim 3, wherein the recombinant expression vector has a cleavage map of FIG. 2B. A host cell transformed with the recombinant expression vector of claim 3. 7. The transformed host cell of claim 6, wherein said host cell is a CHO cell. An immune cell proliferation inhibitor comprising the rhesus monkey CTLA-4 fusion immunoglobulin of claim 1 as an active ingredient. 9. The immune cell proliferation inhibitor according to claim 8, wherein the immune cell is suppressed by blocking an allogeneic response by the fusion immunoglobulin. A method for inhibiting immune cell proliferation comprising treating an immune cell with rhesus monkey CTLA-4 fusion immunoglobulin according to claim 1 in vitro.

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