KR101103603B1 - A method for isolating and expanding antigen-specific CD8+ T cells using an 4-1BB ligand pentamer protein - Google Patents

Abstract

The present invention relates to a method for isolating and propagating antigen-specific CD8 T cells expressing 4-1BB using 4-1BB ligand pentamer protein, and more specifically, the present invention uses COMP-4-1BBL protein. To isolate and proliferate antigen specific CD8 + T cells.

COMP-h4-1BBL pentamers provided by the present invention not only exhibit similar or greater activity than agonistic anti-4-1BB antibodies, but also antigen specific CD8 + T cells developed using anti-4-1BB antibodies. The isolation and proliferation effects of the same level can be replaced, and since the mass-produced antigen-specific CD8 + T cells have the function of removing cancer cells and cells infected with viruses, the present invention has been successful in treating viral diseases and cancer patients. Can be applied.

COMP-4-1BB, CD8 + T cells, separation method, proliferation method

Description

A method for isolating and expanding antigen-specific CD8 + T cells using an 4-1BB ligand pentamer protein}

The present invention relates to a method for isolating and proliferating antigen-specific CD8 + T cells using 4-1BBL pentamer protein.

Attempts to treat cancer using the patient's own immune system have been studied for a long time, and the main targets for cancer immunotherapy are dendritic cells and CD8 + T cells (O'Neill DW, Adams S, Bhardwaj). N. Manipulating dendritic cell biology for the active immunotherapy of cancer.Blood. 2004. 104: 2235-2246 .; Xu D, Gu P, Pan PY, Li Q, Sato AI, Chen SH.NK and CD8 + T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation.Int J Cancer. 2004. 109: 499-506 .; Freeman GJ, Sharpe AH, Kuchroo VK.Protect the killer: CTLs need defenses against the tumor.Nat Med. 2002. 8: 787-789.). Dendritic cells have been studied as promising immunotherapeutic candidates due to their high induction of immune responses to labeled antigens as potent antigen-presenting cells (O'Neill DW, Adams S, Bhardwaj N. Manipulating dendritic cell biology for the active immunotherapy of cancer.Blood. 2004. 104: 2235-2246 .; Preynat-Seauve O, Schuler P, Contassot E, Beermann F, Huard B, French LE.Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection.J Immunol. 2006. 176: 61-67.). However, recent reports have reported that due to the functional diversity of dendritic cells, not only the immunostimulating effect expected in vivo but also immunosuppressive effects (Zhang X, Huang H, Yuan J, Sun D, Hou). WS, Gordon J, Xiang J. CD4-8- dendritic cells prime CD4 + T regulatory 1 cells to suppress antitumor immunity.J Immunol. 2005. 175: 2931-2937 .; Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, Messina JL, Chandler P, Koni PA, Mellor AL.Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes.J Clin Invest. 2004. 114: 280-290. Cancer treatment using dendritic cells is also ultimately aimed at potent activity of CD8 + T cells, and induced CD8 + T cell anticancer reactions are known to inhibit cancer cell recurrence by forming immune T cells. .

In general, antigen-specific CD8 + T cells are isolated using MHC class I / peptide multimers (Valmori D, Pittet MJ, Rimoldi D, LiD, Dunbar R, Cerundolo V, Lejeune F, Cerottini JC, Romero P. An antigen-). targeted approach to adoptive transfer therapy of cancer.Cancer Res. 1999. 59: 2167-73.), this method has a high rate of death by apoptosis after cell separation to produce a sufficient amount of antigen specific CD8 + T cells. There was a drawback to long-term culture. Therefore, surrogate markers were required to separate TCR-stimulating MHC multimers to isolate antigen-specific CD8 + T cells.

4-1BB is expressed in T cells activated as inducible co-stimulatory molecules, and not only enhances the activity of CD8 + T cells but also expresses anti-apoptotic molecules such as Bcl-2, Bcl-XL and Bfl-1. It is well known to show its ability to inhibit activation-induced cell death (AICD) by increasing its activity (Kim YH, Seo SK, Choi BK, Kang WJ, Kim CH, Lee SK, Kwon BS. 4-1BB costimulation enhances HSV-1-specific CD8 + T cell responses by the induction of CD11c + CD8 + T cells.Cell Immunol. 2005. 238: 76-86 .; Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW , Brown TJ, Emswiler J, Raecho H, Larsen CP, Pearson TC, Ledbetter JA, Aruffo A, Mittler RS. 4-1BB costimulatory signals preferentially induce CD8 + T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.J Exp Med. 1997. 186: 47-55 .; Cannons JL, Lau P, Ghumman B, DeBenedette MA, Yagita H, Okumura K, Watts TH.4-1BB ligand induces cel division, sustains survival, and enhances effector function of CD4 and CD8 + T cells with similar efficacy.J Immunol. 2001. 167: 1313-1324 .; Hurtado JC, Kim YJ, Kwon BS. Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J Immunol. 1997.158: 2600-2609. Therefore, the inventors have established a method for isolation and proliferation of antigen-specific CD8 + T cells using an anti-4-1BB antibody using 4-1BB expression of antigen-specific activated CD8 + T cells. However, in the case of antibodies, the half-life of in vitro and in vivo is long, and the effect of the antibody is shown by attaching and acting on the Fc receptor. Will appear. In addition, there are many cases in which various antibodies exist for the same antigen, and they are slightly different from each other.

As a method to replace the problems of these antibodies has been proposed using a 4-1BB ligand protein. However, monomeric recombinant 4-1BB and 4-1BB ligand proteins have low affinity, and to overcome this, natural 4-1BB and 4-1BB ligand proteins are known to bind in the form of trimer (trimer) ( Idriss HT, Naismith JH.TNF alpha and the TNF receptor superfamily: structure-function relationship (s) .Microsc Res Tech. 50 (3): 184-95. 2000.).

In the present invention, a dimer-forming eukaryotic transcriptional activator protein (Heuck A, Du TG, Jellbauer S, Richter K, Kruse C, Jaklin S, MM, Buchner J, Jansen RP, Niessing D. Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes.Proc Natl Acad Sci US A. 104: 19778-83. 2007.), MAT-1 (Matrilin-1) (Wu JJ, Eyre DR.Matrilin-3 forms disulfide -linked oligomers with matrilin-1 in bovine epiphyseal cartilage.J Biol Chem. 273 (28): 17433-8.1998. , Schulthess T, Engel J. The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel Science. 274: 761-5. 1996.) of the human-derived 4-1BB ligand protein at the N-terminal of the protein By combining and expressing the extracellular domain, 4-1BB ligands in the form of dimer, trimer, and pentamer were prepared. T cell activating function and antigen specific CD8 + T cell separation / proliferation ability were investigated. The oligomer, COMP-4-1BBL, showed the strongest T cell proliferative capacity, and was produced successfully using the produced COMP-4-1BBL protein. The present invention has been accomplished by isolating and propagating antigen specific CD8 + T cells.

The present invention is to provide a method for isolating and proliferating antigen-specific CD8 + T cells using the 4-1BB ligand pentamer protein.

In order to achieve the above object, in the present invention, GCN4 (eukaryotic transcriptional activator protein) forming a dimer, MAT-1 (Matrilin-1) constituting a terpolymer, and COMP (cartilage oligomeric matrix protein constituting an oligomer) ) 4-1BB ligand in the form of dimer, terpolymer, or oligomer was prepared by binding and expressing the extracellular domain of human-derived 4-1BB ligand protein at the N-terminus of the protein, and their T cell activity rate and antigen specificity. By examining the rate of intracellular signaling and the isolation and proliferation of antigen-specific CD8 + T cells, the oligomeric form of COMP-4-1BBL showed similar or higher levels of CD8 + T cell proliferation than anti-4-1BB antibodies. It was confirmed that Th1 type cytokine expression is also excellent.

In addition, in the present invention, in order to demonstrate that it is possible to apply various antigen-specific CD8 + T cells in isolation using COMP-4-1BBL instead of the anti-4-1BB antibody, Cytomegalovirus (CMV) pp65 specific in human blood After separating and propagating enemy CD8 + T cells, CMV pp65 / MHC multimer was used to measure the purity of the proliferated cells was confirmed to have an excellent effect.

Thus, in the present invention, it is effective to isolate antigen-specific CD8 + T cells using recombinant protein multimers, not antibodies, and to propagate 4-1BB signals to CD8 + T cells during separation. It was confirmed that can be promoted.

Hereinafter, the present invention will be described in detail.

According to the first aspect of the present invention,

The present invention relates to a method for isolating antigen specific CD8 + T cells from blood samples using binding to a cargo oligomeric matrix protein (COM) -4-1BB ligand protein.

In the present invention, preferably, the antigen-specific CD8 + T cells are isolated by culturing together PBMCs (peripheral blood mononuclear cells) isolated from the blood by adding the antigen peptides in a culture medium to be specific for the antigen peptides. First step to form CTL (CD8 + T lymphocyte); Incubating the culture of step 1 with the COMP-h4-1BB ligand to bind the peptide specific CTL and COMP-h4-1BB ligand; And a third step of separating only the cells specifically bound to the COMP-h4-1BB ligand from the culture of the second step.

In the present invention, the antigenic peptide of the first step may be derived from a virus or a cancer antigen. Preferably, the virus-derived peptide is selected from a CMV (Cytomegalovirus) peptide, an EBV (Epstein-barr Virus) peptide, and a human telomerase reverse transcriptase (hTERT) peptide. Also preferably, the cancer antigen-derived peptide may be a WT-1 (Wilm's Tumor-associated gene) peptide.

In the present invention, the culture medium of the first step is preferably a medium containing autologous plasma or autologous serum, and human IL-2. In addition, the first step of the culture is preferably cultured for 12 days to 16 days. In addition, the second step of the culture is characterized in that the culture for 1 to 3 hours.

In one embodiment of the present invention, in connection with the first step, after separating the PBMC from the blood of CMV pp65 pentamer positive volunteers, and dispensed in a 15ml tube at a concentration of 2x10 ⁶ cells / ml, and then at a concentration of 1ug / ml CMV pp65 peptide (NLVPMVATV) was added. After 48 hours of culture, a medium containing 50 IU / ml recombinant human IL-2 (Proleukin) was added to promote cell proliferation. After removal of 1 ml of medium from day 7 of cell culture, 1 ml of medium containing fresh IL-2 was added again, and this was repeated at two-day intervals. At 14 days of culture, cells were harvested, and CMV pp65-specific CD8 + T cells were incubated by adding 1 μg / ml of CMV pp65 peptide, and after 24 hours, cells were harvested and analyzed by flow cytometry. Expression of -1BB was confirmed.

In addition, in one embodiment of the present invention, in relation to the second step and the third step, after washing the culture plate coated with COMP-h4-1BBL at a concentration of 10ug / ml for 5 days with PBS, 4-1BB The cells expressing the cells were added and incubated for one hour in a 37 ° C. CO ₂ incubator. After incubation, the culture plate was gently washed with PBS to isolate only the cells specifically bound to COMP-h4-1BBL.

The isolated cells were harvested and flow cytometrically analyzed for CMV pp65 specificity of cells isolated by anti-4-1BB antibody or by COMP-h4-1BBL, both of which were CMV pp65-specific CD8 + T cells with at least 95% purity. It was confirmed that was separated (Fig. 3A).

Furthermore, according to the second aspect of the present invention,

The present invention relates to a method of mass proliferating antigen-specific CD8 + T cells isolated by the above method. Preferably, in the present invention, the proliferation is characterized in that the culture in a medium containing IL-2. In addition, the culture is characterized in that 14 to 28 days to culture.

In one embodiment of the present invention, isolated CMV pp65-specific CD8 + T cells were proliferated for 2 weeks in IL-2 conditions at a concentration of 1000 IU / ml. When the IFN-γ expression level was measured by reactivating the proliferated CD8 + T cells with CMV pp65 peptide at 28 days of culture, more than 80% of the CD8 + T cells isolated and expanded under two conditions express high levels of IFN-γ. Was confirmed (FIG. 3B). In addition, in order to measure the cytotoxicity of CD8 + T cells, the expression rate of CD107a (LAMP-1) expressed on the cell surface after CMV pp65 peptide treatment was measured. As a result, the cytotoxicity was found in 50% or more cells (FIG. 3B). ).

One embodiment of the present invention to compare the effect of using an anti-4-1BB antibody with the method for isolating or propagating antigen-specific CD8 + T cells using the COMP-h4-1BB ligand protein of the present invention in, wherein a result of the same experiment using a -4-1BB antibody, when using PBMC isolated from 30ml of blood after the final incubation hayeoteul count the number of 28 days of cell proliferation was about 5 - 6 x 10 ⁷ cells, level And both anti-4-1BB antibody and COMP-h4-1BBL had similar proportions (FIG. 3C). In addition, when the number of proliferated cells was converted into an increase rate, both showed an increase rate of about 1250 times. That is, these results demonstrate that the recombinant protein COMP-h4-1BBL can be used in place of the anti-4-1BB antibody.

As described above, antigen-specific CD8 + T cells can be effectively separated using recombinant protein multimers according to the present invention, and 4-1BB signal is transmitted to CD8 + T cells during the isolation process to promote the proliferation of the isolated cells. can do.

COMP-h4-1BBL pentamers constructed and validated in the present invention exhibit similar or higher activity than agonistic anti-4-1BB antibodies, as well as antigen specific CD8 + T developed with anti-4-1BB antibodies. It has been confirmed that the separation and proliferation effects of the cells can be replaced at the same level. Since the mass-produced antigen-specific CD8 + T cells have a function of removing cancer cells and cells infected with the virus, the viral disease is prevented by the present invention. And it is expected to be successfully applied to the treatment of cancer patients.

Hereinafter, the present invention will be described in detail by the following examples. However, the following Examples and Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples.

Example 1 Gene Construct and Cloning

4-1BBL is one of the TNFSF (TNF superfamily), which is known to form a trimer and bind to the receptor 4-1BB (Rabu C, QuA, Jacques Y, Echasserieau K, Vusio P, Lang F. Production of recombinant human trimeric CD137L (4-1BBL) .Cross-linking is essential to its T cell co-stimulation activity.J Biol Chem. 280: 41472-81. 2005.). Therefore, in the present embodiment, 4-1BB signaling and isolation of 4-1BB + CD8 + T cells by constructing the 4-1BBL extracellular domain in the form of dimers, trimers, and pentamers for the purpose of replacing anti-4-1BB antibodies. 4-1BBL polypolymer fabrication was performed in a form suitable for.

That is, in the present embodiment, GCN4-h4-1BBL, MAT-1-h4-1BBL, and COMP-h4-1BBL in pD18 (including CD5L-FLAG tag), a plasmid vector for expressing CHO cell line proteins, as BamH I / Not I sites. Cloned. Since GCN4 forms dimers, MAT-1 forms trimers, and COMP forms pentamers, it was expected that h4-1BBL expressed in combination with these proteins would also form dimers, trimers, pentamers (Fig. 1A). Each DNA construct was transfected into CHO cells, and CHO cell lines expressing high levels of h4-1BBL were selected by gradually increasing the concentration of MTX. Proteins were produced through mass culture, and an affinity column immobilized with anti-h4-1BBL mAb was prepared to separate each protein.

1-1 Gene Construct

First, in the pD18 vector with the signal sequences CD5L and FLAG-tag, human 4-1BBL extracellular domain and yeast GCN4, chicken matrilin 1 (MAT-1), or human cartilage oligomeric matrix protein (COMP) Cloned (Table 1).

Amino Acid Sequences and DNA Sequences of CD5L, FLAG, h4-1BBL, GCN4, hMAT-1, and hCOMP gene Amino acid sequence
(SEQ ID NO) DNA sequence
(SEQ ID NO) CD5L MPMGSLQPLATLYLLGMLVASV
(SEQ ID NO 1)
ATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTG
(SEQ ID NO: 2) FLAG DYKDDDDK
(SEQ ID NO: 3) GACTACAAGGACGACGATGACAAG
(SEQ ID NO: 4) h4-1BBL SPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVL
(SEQ ID NO: 5)



(SEQ ID NO: 6) GCN4 MKQLEDKVEELLSKNYHLENEVARLKKLVGE
(SEQ ID NO: 7)
ATGAAACAACTTGAAGACAAGGTTGAAGAATTGCTTTCGAAAAATTATCACTTGGAAAATGAGGTTGCCAGATTAAAGAAATTAGTTGGCGAA
(SEQ ID NO: 8) hMAT-1 EEDPCACESLVKFQAKVEGLLQALTRKLEAVSKRLAILENTVV
(SEQ ID NO: 9)
GAGGAAGACCCGTGTGCCTGCGAGTCCCTGGTGAAATTCCAAGCCAAAGTGGAGGGGCTGCTGCAGGCCCTGACCAGGAAACTGGAAGCTGTGAGTAAGCGGCTGGCCATCCTGGAGAACACAGTTGTC
(SEQ ID NO: 10) hCOMP DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACG
(SEQ ID NO: 11) GACCTGGGCCCGCAGATGCTTCGGGAACTGCAGGAAACCAACGCGGCGCTGCAGGACGTGCGGGAGCTGCTGCGGCAGCAGGTCAGGGAGATCACGTTCCTGAAAAACACGGTGATGGAGTGTGACGCGTGCGGG
(SEQ ID NO: 12)

1-2 cloning

1-2-1 PCR reaction

PCR was pre-denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, and annealing varied with temperature depending on the gene, but the basic time was 30 seconds, and elongation was 30 seconds at 72 ° C. Elongation was carried out at 72 ° C. for 7 minutes under post-elongation conditions.

The sequence of primers used for each gene amplification is as follows.

GCN4 F: GGATCCATGAAACAACTTGAAGAC (24mer)-BamHI (SEQ ID NO: 13)

        R: GAATTCTTCGCCAACTAATTTCTT (24mer)-EcoRI (SEQ ID NO: 14)

hMAT1 F: GGATTCGAGGAAGACCCGTGTGCC (24mer)-bamHI (SEQ ID NO: 15)

        R: GAATTCGACAACTGTGTTCTCCAG (24mer)-EcoRI (SEQ ID NO: 16)

hCOMP F: GGATCCGACCTGGGCCCGCAGATG (24mer)-BamHI (SEQ ID NO: 17)

        R: GAATTCCCCGCACGCGTCACACTC (24mer)-EcoRI (SEQ ID NO: 18)

h4-1BBL F: GAATTCAGCCCGAGACTCCGCGAG (24mer)-EcoRI (SEQ ID NO: 19)

        R: GCGGCCGCTTATGGGATTTCGGGGGT (26mer)-Not I (SEQ ID NO: 20)

In order to amplify the recombinant 4-1BBL gene including the 4-1BBL extracellular domain, the above primers were used to amplify 35 cycles at 56 ° C, 30 cycles at 40 ° C annealing conditions, and MAT-1 at 45 ° C annealing. 30 cycles under conditions, hCOMP amplified the gene for 30 cycles under 60 ℃ annealing conditions.

Cloning into T vector

Each PCR product was mixed with a 3: 1 ratio of the insert and the vector for ligation to the pGEM T easy vector (promega), incubated at 16 ° C. for 2 hours with the addition of ligase (promega) and 2X buffer. It was. Regates were transformed into DH5α competent cells (using a standard molecular method), followed by X-gal and IPTG color selection to select only positive colonies. Each final clone was selected by sequencing the final selected clones to confirm their sequencing.

Cloning into pD18 vector

T vector clones containing GCN4, MAT-1, COMP were digested with BamH I / EcoR I and T vector clones containing h4-1BBL with EcoR I / Not I for one hour to prepare individual gene fragments. At the same time, the pD18 vector containing CD5L-FLAG was digested with BamH I / Not I for one hour to prepare a vector fragment. Insert and the vector were mixed in a 3: 1 ratio, and ligase (promega) and 2X buffer were added and incubated at 16 ° C for 2 hours. Regates were transformed into DH5α competent cells (using standard molecular methods), and colonies were formed. Each clone was inoculated in LB-Amp medium to incubate each clone.

Plasmid DNA was isolated from the cultures using Alkalline lysis method (using solutions I, II, III and isopropanol, 70% ETOH, RNase), and digested with BamH I / Not I for one hour to obtain DNA fragments of the expected size. Confirmed. Positive clones were sequenced to select the final clones, each named pD18-GCN4-h4-1BBL, pD18-MAT1-h4-1BBL, pD18-COMP-h4-1BBL.

Example 2 Selection of CHO Cells Expressing GCN4-h4-1BBL, MAT1-h4-1BBL, COMP-h4-1BBL

2-1 Infection

Chinese hamster ovary (CHO) cell line was added to CHO 302 Medium (JRH, USA) by adding 1 mM sodium pyruvate, recombinant insulin (Recombulin.TM.Zn, Gibco-BRL) 0.5 μg / ml, 2 mM L-glutamine, 1X HT It was used, and incubated in 5% CO _2/37 ℃ incubator.

pD18-GCN4-h4-1BBL, pD18-MAT1-h4-1BBL, pD18-COMP-h4-1BBL DNA were eletroporated in 5X10 ⁶ DHFR-deficient CHO (DHFR-CHO) cells at 500 V, 825 kV (Bio-Rad) . Electroporated cells were suspended in 50 ml CHO cell medium (1 mM sodium pyruvate, 0.5 μg / ml recombulin, 2 mM L-glutamine) containing 20 nM MTX, and 100 μl were dispensed into 96 well tissue culture plates. . After incubation for 10 days in a 37 ℃ CO ₂ incubator was further cultured for 4 days by adding 100 μl of 20nM CHO 302 medium.

2-2 Screening

An enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of 4-1BBL multimer protein using CHO cells. Anti-4-1BBL mAb (5G11; Immunomes Co., Ltd.) was prepared at a concentration of 2 μg / ml in coating buffer (PBS, pH7.4), and then per well in a 96 well plate (Nunc Brand product) for ELISA. Dispense 100 μl and coat at 4 ° C. for one day. 100 μl of blocking buffer (4% BSA in PBS) was aliquoted and blocked for 2 hours at 37 ° C. 200 μl aliquots of PBS-T (0.05% Tween20 in PBS) were washed twice. The culture supernatant containing h4-1BBL multimer protein was reacted at 37 ° C. for 1 hour at 100 μl / well. After 200 μl of PBS-T was washed four times, 100 μl of anti-4-1BBL mAb (4H3-biotin, Immunix Co., Ltd.) was dispensed per well to react at 37 ° C. for 1 hour. 200 μl of PBS-T was washed four times, and 100 μl of HRP-straptavidin (R & D systems) diluted in PBS at 1/2000 was dispensed per well and reacted at 37 ° C. for 1 hour. After washing the plate four times with 200 μl / well of PBS-T, the absorbance was measured at 405 nm by color development for 30 minutes using the ABTS system. CHO cells of the wells with the highest absorbance were dispensed in 96 well plates, and then the MTX concentration was increased to 50 nM, and then the MTX concentration was 100 nM, 200 nM, 500 nM, and 1000. The clones were raised to nM and repeatedly repeated to produce the highest levels of m4-1BBL multimers.

2-3 Mass Production with Wave Bioreactor

200 ml of CHO 302 cell medium and 7X10 ⁵ cells / ml of selected clones (GCN4-, MAT-1-, COMP-h4-1BBL) were incubated in a cell bag (rocking speed: 17 rpm, CO ₂ : 10%, angle: 7, temperature: 37 ℃). CO ₂ was reduced to 5% at 24 hours of incubation and sampled daily to determine pH and number of cells. After 2-3 days of culture, 800 ml of CHO 302 medium was added so that the number of cells became 2 × 10 ⁶ cells / ml and the culture volume was 1 L. Then cultured again (rocking speed: 20 rpm, angle: 7.5 remainder). In addition, pH and cell viability were confirmed by daily sampling, and cells were collected when the cell activity was reduced to 40-50%.

2-4 Antibody-Friendly Gel Making with Disuccinimidyl Suberate (DSS)

2 ml of protein G-agarose resin was transferred to the column and washed three times with 3 ml of PBS. 500 μg of anti-4-1BBL mAb (4H3-biotin, Immunomix Co., Ltd.) was adjusted to a total volume of 1 ml in PBS, and filled in a column containing protein G-agarose and incubated at room temperature for 1 hour. . And to remove the unbound Ab 3 ml of PBS was added and carefully suspended and washed, this process was repeated 2-3 times. After the last wash, the suspension was gently suspended with 1 ml PBS, and 50 µl (0.625 mg) of 13 mg / ml DSS (sigma) was added, followed by careful suspension and incubation at room temperature for 1 hour. Wash 5 times with 3 ml of 0.1 M glycine (pH 2.5) to remove DSS and uncoupled Ab. Again washed twice with 3 ml PBS and used for protein isolation.

2-5 Affinity purification

The h4-1BBL polypolymer protein was purified using the 4H3-protein G affinity gel prepared in Example 3-4. After washing the affinity gel with PBS, the culture supernatant containing h4-1BBL polypolymer was passed through the column. After washing the column three times with PBS 0.1M glycine (pH2.5) was added to elute the protein. The isolated protein was quantified after PBS dialysis and used in the next experiment.

2-6 Identification of Isolated Proteins

In addition, in order to confirm the size and mutimerization of each of the separated proteins, the protein was prepared using a sample buffer solution containing 2-mercaptoethanol (2-ME) and a sample buffer solution not included. Electrophoresis performed western blot with anti-4-1BBL antibody.

In the case of GCN4-h4-1BBL, which is expected to form a dimer, 26 KDa size was observed when 2-ME was added, and when the multimer was formed without 2-ME addition, it was 4 at 52 KDa. -1BBL was detected (FIG. 1B). Thus, GCN4-h4-1BBL was found to form dimers as expected.

In the case of MAT-1-h4-1BBL, the size of the monomer was 27.8 KDa, and in the case of the multimer, it was confirmed that a trimer was formed by detecting at a size of 83 KDa (FIG. 1C).

In the case of COMP-h4-1BBL, the monomer was about 28 KDa in size, and in the multimer, it was detected at about 140 KDa in size. Thus, COMP-h4-1BBL was found to form pentamers (FIG. 1D).

These results demonstrate that each construct normally formed dimers, trimmers, and pentamers.

Example 3 Proliferation assay

In this Example, to investigate whether the growth rate of human CD8 + T cells by 4-1BBL dimer, trimer, pentamer was increased, the h4-1BBL dimer, trimer, and pentamer separated and purified in Example 2 were separated. CD8 + T cells were treated to measure changes in CD8 + T cell proliferation.

3-1 Isolation of CD8 + T Cells

30 ml of blood was collected from volunteers, and 7 ml of blood was gently dispensed into the upper 15 ml cornical tube containing 7 ml of Ficoll-hypaque. The tube was centrifuged at 2000 rpm for 20 minutes at room temperature, and PBMC (peripheral blood mononuclear cell) of the middle layer was separated. The isolated PBMCs were washed three times with PBS. In order to separate CD8 + T cells, PBMC and CD8-microbead (Miltenyi Biotec) were mixed and incubated at 4 ° C. for 30 minutes, and the cells were passed through LS column (Miltenyi Biotec) to separate only CD8 + T cells.

3-2 Measurement of Changes in CD8 + T Cell Proliferation

(1) After dispensing the isolated CD8 + T cells in 96-well plates at a concentration of 2x10 ⁵ / well, CD8 + T cells were activated by treatment with an anti-CD3 antibody at a concentration of 0.05 ug / ml, and simultaneously GCN4-h4-1BBL, Changes in CD8 + T cell proliferation were measured by treatment of MAT-1-h4-1BBL and COMP-h4-1BBL at 120 ng / ml or 240 ng / ml. As a control, two kinds of agonistic anti-4-1BB mAb (4B4, 20.G4) were used at a concentration of 5 ug / ml. Cells were incubated for 3 days and treated with [ ³ H] -thymidine at 1.0 uCi / well concentration for the last 8 hours of culture to examine cell proliferation.

CD8 + T cells treated with anti-CD3 antibody showed proliferation of about 10,000 cpm, and 1.5-fold increase in anti-4-1BB antibody 4B4 and 2.5 increase in 20.G4 treatment. However, when GCN4-h4-1BBL treatment did not change at 120ng / ml concentration, treatment at 240ng / ml concentration showed a 1.3-fold proliferation rate. MAT-1-h4-1BBL treatment did not show a significant change in both 120ng / ml, 240ng / ml concentration. However, COMP-h4-1BBL treatment showed an increase of about 2 to 2.5 times in cell proliferation at both 120ng / ml and 240ng / ml concentrations (FIG. 2A). These results demonstrate that the pentamer type h4-1BBL effectively proliferates CD8 + T cells compared to dimers or trimers.

(2) In addition, in order to more accurately verify that the CD8 + T cell proliferation promoting effect by GCN4-, MAT-1-, COMP-4-1BBL was caused by the 4-1BBL protein portion, an anti-4-1BBL antibody was used. The same experiment was conducted after neutralizing the 4-1BBL portion of each 4-1BBL multimer. CD8 + T cells treated with Anti-CD3 antibody (0.05ug / ml) were pre-incubated with 240ng / ml GCN4-, MAT-1-, COMP-4-1BBL or 5ug / ml anti-4-1BBL antibody After treatment with GCN4-, MAT-1-, COMP-4-1BBL, and incubated for 72 hours, cell proliferation rate was measured by labeling with [3H] -thymidine for the last 8 hours. In these results, COMP-h4-1BBL most effectively induced the proliferation of CD8 + T cells, and each h4-1BBL multimer reduced the CD8 + T cell proliferation rate when pre-incubated with the anti-4-1BBL antibody. 2B). Thus, these results demonstrate that the pentamer form COMP-h4-1BBL most effectively proliferates CD8 + T cells among the three h4-1BBL multimers, and this effect was induced by h4-1BBL.

Example 5 Cytokine Assay

The effect of Th1 / 2 cytokine expression on CBA kit was investigated from the cell culture supernatant cultured for 48 hours. IFN-γ, IL-2, IL-10, IL-4, IL-5, TNF- in supernatant of CD8 + T cells treated with anti-CD3 antibody and h4-1BBL multimer or anti-4-1BB antibody (4B4) The concentration of α was measured by flow cytometry.

That is, CD8 + T cells isolated through the same procedure as in Example 4 were dispensed into 96 well plates at a concentration of 2 × 10 ⁵ / well, anti-CD3 antibody at 0.05 ug / ml, and anti-4-1BB antibody at 5 ug. At / ml concentrations, various h4-1BBL polypolymer proteins were treated at 120 or 240 ng / ml concentrations. The culture supernatant was prepared by culturing the cells for two days, and the amount of each cytokine was measured by flow cytometry using a CBA kit (BD Bioscience) and 50 ul of the culture supernatant.

As a result, the expression of TNF-α was increased by anti-4-1BB antibody treatment, but the expression of IFN-γ was significantly increased by COMP-4-1BBL treatment. When treated with GCN4- or MAT-1-h4-1BBL, the expression of TNF-α was significantly increased rather than IFN-γ similarly to the anti-4-1BB antibody (FIG. 2C). This result demonstrates that the COMP-h4-1BBL protein increases the expression of IFN-γ, a representative Th1-type cytokine.

Example 6 Isolation of Antigen-Specific CD8 + T Cells

6-1 Isolation and Proliferation of Antigen-specific CD8 + T Cells Using COMP-h4-1BBL

1) Introduction and proliferation of CMV peptide specific CTLs

1. Collect peripheral blood (30 ml) using heparin from a HLA-A * 02 positive (do not use EDTA or citric acid).

2. Dispense 7ml of blood into 15ml cornical tube, put 7ml Ficoll (1.077) at the bottom of the blood to induce layer separation according to the density, and centrifuge at 900 x g for 20 minutes.

3. Collect autoplasma and PBMC formed after centrifugation in order. Autologous plasma should be stored at 4 ° C until use.

4. The collected PBMCs are washed by centrifugation for 5 minutes at 1200 rpm with 50 ml of RPMI1640 medium. Repeat this process twice.

5. Suspend the cells in RPMI1640 medium containing 5% autologous plasma. Count the cells and adjust to 2 x 10 ⁶ cells / ml.

6. Add 1 ml / ml of CMV pp65 peptide (NLVPMVATV) to the suspended PBMC. Incubate 1ml into 15ml tube.

CMV pp65 (495-504 amino acid, NLVPMVATV); HLA-A * 0201-restricted

7. On the second day of culture (PI day 2), add 10 ml of RPMI1640 medium containing 5% autologous plasma and 50 IU / ml human IL-2.

8. On day 7 of culture (PI day 7), remove 1 ml of cell culture medium and add 1 ml of RPMI1640 medium containing 5% autologous plasma and 50 IU / ml human IL-2.

9. Remove 1 ml of the culture every two days and add 1 ml of RPMI1640 medium containing 5% autologous plasma and 50 IU / ml human IL-2 (PI day 9, 11, 13).

10. Formation of CMV-specific CTLs on day 14 of culture is confirmed using MHC pentamers.

-CMV pp65 NLVPMVATV (Proimmune, HLA-A * 0201)

   2) Reactivation of CMV Peptide Specific CD8 + T Cells

1. Incubate in a T-25 flask by adding 1 ug / ml peptide to the CTL prepared in the above procedure. The medium is X-VIVO 10 medium containing 50 IU / ml IL-2 and 5% autologous cells, and incubated at a concentration of 2 x 10 ⁶ cells / 1ml / well.

2. Incubate for 24 hours in a 37 ° C. CO ₂ incubator.

3. Two days after culture, the ratio of 4-1BB positive cells in CD8 ⁺ T cells was analyzed by flow cytometry using PE-conjugated anti-4-1BB antibody.

PE-conjugated anti-4-1BB mAb; BD Pharmingen

FITC-conjugated anti-CD8 + mAb; BD Pharmingen

That is, as described above, after separating PBMC from the blood of CMV pp65 pentamer positive volunteers, the cells were dispensed into a 15ml tube at a concentration of 2x10 ⁶ cells / ml, and then CMV pp65 peptide (NLVPMVATV) was added at a concentration of 1ug / ml. After 48 hours of culture, a medium containing 50 IU / ml recombinant human IL-2 (Proleukin) was added to promote cell proliferation. After removal of 1 ml of medium from day 7 of cell culture, 1 ml of medium containing fresh IL-2 was added again, and this was repeated at two-day intervals. On day 14 of culture, cells were harvested and reactivation of CMV pp65-specific CD8 + T cells was induced by culturing with addition of CMP pp65 peptide at a concentration of 1 ug / ml. After 24 hours, cells were harvested and confirmed 4-1BB expression by flow cytometry.

After washing the culture plate coated with COMP-h4-1BBL or anti-4-1BB antibody at a concentration of 10 ug / ml for 5 days with PBS, and then adding cells expressing 4-1BB for one hour in a 37 ° C CO2 incubator. Incubated for 2 hours. After incubation, the culture plate was gently washed with PBS to isolate only the cells specifically bound to the anti-4-1BB antibody or COMP-h4-1BBL.

The isolated cells were harvested and flow cytometrically analyzed for CMV pp65 specificity of cells isolated by anti-4-1BB antibody or by COMP-h4-1BBL, both of which were CMV pp65-specific CD8 + T cells with> 95% purity. Was isolated (FIG. 3A).

Isolated CMV pp65-specific CD8 + T cells were proliferated for 2 weeks under IL-2 conditions at 1000 IU / ml. When the IFN-γ expression level was measured by reactivating the proliferated CD8 + T cells with CMV pp65 peptide at 28 days of culture, at least 80% of the CD8 + T cells isolated and expanded under two conditions expressed high levels of IFN-γ. (FIG. 3B).

In addition, in order to measure the cytotoxicity of CD8 + T cells, the expression rate of CD107a (LAMP-1) expressed on the cell surface after CMV pp65 peptide treatment was measured. As a result, the cytotoxicity was found in 50% or more cells (FIG. 3B). ).

6-2 Isolation and Proliferation of Antigen-specific CD8 + T Cells Using Anti-4-1BB Antibody

On the other hand, as a comparative experiment for Example 6-1, the same process was performed for the anti-4-1BB antibody. The sequence of the comparative experiment is shown as follows.

1) 4-1BB ⁺ CD8 ⁺  Isolation and Mass Propagation of T Cells

1. Dispense anti-4-1BB antibody (4B4) or COMP-h4-1BBL protein diluted in PBS at a concentration of 10ug / ml into 10ml / flask and store at 4 ° C for 20-24 hours.

2. After storage, the supernatant containing the antibody is removed, and the BSA solution dissolved at 2.5% concentration in PBS is dispensed with 10ml / flask and stored at 4 ° C for 20-24 hours without washing.

3. After removing the BSA solution, wash each flask twice with 15 ml PBS.

4. The cells prepared in the above procedure are suspended in X-VIVO 10 medium and dispensed into each flask coated with humanized anti-4-1BB antibody and incubated for 1 hour in a 37 ° C CO ₂ incubator.

5. Remove the supernatant after incubation for 1 hour and wash twice with 10ml RPMI1640 medium.

6. Add X-VIVO 10 medium containing 2% autologous plasma and 1000 IU / ml IL-2 to each flask and incubate for 14 days.

   2) Flow cytometry

In order to prevent nonspecific binding of the chromosome antibodies through the Fc region, 10 μl of 1 mg / ml human IgG solution was mixed with the cells and reacted at 4 ° C. for 10 minutes, followed by 30 minutes at 4 ° C. by adding an antibody specific to surface molecules. By staining. Cells stained with each antibody were washed twice with PBS containing 0.1% BSA, and the prepared samples were analyzed for surface molecular phenotype using flow cytometry (BD Bioscience, San Diego, Calif.).

The cell surface exposure of CD107α was measured to investigate antigen-specific CTL function. Cells to be tested for CTL ability were dispensed into 96 well plates at a concentration of 2 × 10 ⁶ cells / well, and then CMV peptides were treated at a concentration of 5 ug / ml. At the same time 1 μl of 2 mM monensin (monensin, Sigma) and anti-CD107a-biotin antibody were added. The cells were mixed in a multichannel pipette, the plates were centrifuged at 300 xg for 1 minute and the cells were allowed to settle and incubated at 37 ° C for 5 hours. After incubation, cells were harvested from each well and cells were disrupted by washing the cells with PBS containing 0.02% azide and 0.5 mM EDTA. Samples were stained with streptavidin-FITC, anti-CD8-PE-Cy5 and peptamer-PE for 30 minutes. All stained cells were analyzed by flow cytometry.

Compared with the results of the comparative experiment, after the final culture, the number of cells proliferated for 28 days using PBMC isolated from 30 ml of blood was about 5-6x107 cells, and anti-4-1BB antibody and COMP All of the -h4-1BBLs had a similar ratio (FIG. 3C). In addition, when the number of proliferated cells was converted into an increase rate, both showed an increase rate of about 1250 times.

That is, these results demonstrate that the recombinant protein COMP-h4-1BBL can be used in place of the anti-4-1BB antibody.

As described above, the antibody is synthesized by the result of signaling by the Fab fragment attached to the target protein and the signal transduction through the Fc receptor to which the Fc fragment binds, and the Fc receptor may increase or decrease the immune response depending on the type. (Nimmerjahn F, Ravetch JV. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 310: 1510-2. 2005.), precisely verifying the effect of antibodies exhibiting bidirectional signaling It is not easy, it may cause unexpected problems, and because of the long half-life of the protein, even if a problem occurs, the side effect may continue to appear. To overcome this problem, there is a need for the development of an agent that represents only authentic signaling and can exhibit effects similar to or better than antibodies. Thus, the COMP-h4-1BBL pentamer constructed and validated in the present invention exhibits similar or greater activity than the agonistic anti-4-1BB antibody, as well as antigen specific developed using an anti-4-1BB antibody. It has been found that the isolation and proliferation effects of CD8 + T cells can be replaced at the same level. Since mass-produced antigen-specific CD8 + T cells have a function of removing cancer cells and cells infected with viruses, It is expected to be successfully applied to the treatment of sexual diseases and cancer patients.

1 shows the production of various h4-1BBL multimers. For the production of h4-1BBL multimers, the h4-1BBL extracellular domain, GCN4, MAT-1, and COMP were combined and cloned into pD18, a CHO cell expression plasmid vector.

(A) Schematic diagram of the gene sequence and restriction enzyme position of each DNA construct and the protein expressed. (B) Purified GCN4-h4-1BBL isolated from CHO cell culture supernatant using (+ 2-ME) sample buffer with 2-ME or (2-ME) sample buffer without 2-ME SDS-PAGE running was carried out to the nitrocellulose membrane and Western blotting was performed using anti-h4-1BBL antibody. (C) Western blot results of MAT-1-h4-1BBL, (D) Western blot results of COMP-h4-1BBL

2 shows effective CD8 + T cell proliferation and Th1 cytokine expression by COMP-h4-1BBL. CD8 + T cells isolated from PBMC were isolated and aliquoted into 96 well plates at 2 × 10 ⁵ concentration. Anti-CD3 antibody (OKT3) was treated at a concentration of 0.05 ug / ml to induce CD8 + T cell proliferation.

(A) Simultaneously treated with GCN4-h4-1BBL (GCN4), MAT-1-h4-1BBL (cMAT), COMP-h4-1BBL (COMP) at 120 ng / ml or 240 ng / ml. 4B4 or 20.G4, 1BB mAb, was treated at a concentration of 5 ug / ml. (B) h4-1BBL multimer and anti-h4-1BBL antibodies, respectively (5G11; 5ug / ml), to neutralize 4-1BBL of GCN4-h4-1BBL, MAT-1-h4-1BBL, COMP-h4-1BBL After mixing, the treated CD8 + T cells activated with anti-CD3 antibody. Cells were incubated for 72 hours in a 37 ° C. CO ₂ incubator and labeled with [ ³ H] -thymidine at 1 uCi / well concentration for the last 8 hours. Radiolabeling degree was determined by liquid scintillation spectroscopy. (C) From the supernatants collected at 48 hours of culture, the concentration of IFN-γ, IL-2, IL-10, IL-4, IL-5, TNF-α was measured using a CBA kit according to the protocol provided by the manufacturer. Measured.

3 shows the isolation and proliferation of antigen specific CD8 + T cells using COMP-h4-1BBL. PBMC was isolated from 30 ml of blood of normal volunteers using Ficoll-hypaque, diluted to a concentration of 2x10 ⁶ cells / ml, and then dispensed into 1 ml of 15 ml tubes. At the same time, CMV pp65 peptide was incubated with 1ug / ml concentration. After 48 hours of culture, a medium containing 50 IU / ml recombinant human IL-2 (Proleukin) was added to promote cell proliferation. After removal of 1 ml of medium from day 7 of cell culture, 1 ml of medium containing fresh IL-2 was added again, and this was repeated at two-day intervals. On day 14 of culture, cells were harvested and reactivation of CMV pp65-specific CD8 + T cells was induced by adding CMP pp65 peptide at a concentration of 1 ug / ml. After 24 hours, cells were harvested and confirmed 4-1BB expression by flow cytometry. 4-1BB expressing cells were dispensed in a culture plate coated with COMP-h4-1BBL or anti-4-1BB antibody and incubated for one hour in a 37 ° C. CO ₂ incubator.

(A) 4-1BB + cells attached to the culture plate were harvested and stained with anti-CD8-FITC and CMV pp65 pentamer-Pe (pCMV) for flow cytometry. (B) The isolated cells were grown using a medium containing 1000 IU / ml IL-2, and after treatment with CMV pp65 peptide on day 28 of culture, anti-CD8-FITC and anti-IFN-γ-PE or anti-CD8 Each expression was flow cytometrically stained with -PE and anti-CD107a-biodm. (C) Count the number of final proliferated cells on day 28 of culture. (D) Proliferation rate of proliferated cells.

<110> NATIONAL CANCER CENTER <120> A method for isolating and expanding antigen-specific CD8 + T          cells using an 4-1BB ligand pentamer protein <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> PRT <213> amino acid sequence for CD5L <400> 1 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly   1 5 10 15 Met Leu Val Ala Ser Val              20 <210> 2 <211> 66 <212> DNA <213> DNA sequence for CD5L <400> 2 atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60 tccgtg 66 <210> 3 <211> 8 <212> PRT <213> amino acid sequence for FLAG <400> 3 Asp Tyr Lys Asp Asp Asp Asp Lys   1 5 <210> 4 <211> 24 <212> DNA <213> DNA sequence for FLAG <400> 4 gactacaagg acgacgatga caag 24 <210> 5 <211> 179 <212> PRT <213> amino acid sequence for h4-1BBL <400> 5 Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala   1 5 10 15 Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln              20 25 30 Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly          35 40 45 Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr      50 55 60 Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln  65 70 75 80 Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser                  85 90 95 Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala             100 105 110 Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn         115 120 125 Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln     130 135 140 Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp 145 150 155 160 Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro                 165 170 175 Glu ile pro             <210> 6 <211> 537 <212> DNA <213> DNA sequence for h4-1BBL <400> 6 agcccgagac tccgcgaggg tcccgagctt tcgcccgacg atcccgccgg cctcttggac 60 ctgcggcagg gcatgtttgc gcagctggtg gcccaaaatg ttctgctgat cgatgggccc 120 ctgagctggt acagtgaccc aggcctggca ggcgtgtccc tgacgggggg cctgagctac 180 aaagaggaca cgaaggagct ggtggtggcc aaggctggag tctactatgt cttctttcaa 240 ctagagctgc ggcgcgtggt ggccggcgag ggctcaggct ccgtttcact tgcgctgcac 300 ctgcagccac tgcgctctgc tgctggggcc gccgccctgg ctttgaccgt ggacctgcca 360 cccgcctcct ccgaggctcg gaactcggcc ttcggtttcc agggccgctt gctgcacctg 420 agtgccggcc agcgcctggg cgtccatctt cacactgagg ccagggcacg ccatgcctgg 480 cagcttaccc agggcgccac agtcttggga ctcttccggg tgacccccga aatccca 537 <210> 7 <211> 31 <212> PRT <213> amino acid sequence for GCN4 <400> 7 Met Lys Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr   1 5 10 15 His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu              20 25 30 <210> 8 <211> 93 <212> DNA <213> DNA sequence for GCN4 <400> 8 atgaaacaac ttgaagacaa ggttgaagaa ttgctttcga aaaattatca cttggaaaat 60 gaggttgcca gattaaagaa attagttggc gaa 93 <210> 9 <211> 43 <212> PRT <213> amino acid sequence for hMAT-1 <400> 9 Glu Glu Asp Pro Cys Ala Cys Glu Ser Leu Val Lys Phe Gln Ala Lys   1 5 10 15 Val Glu Gly Leu Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser              20 25 30 Lys Arg Leu Ala Ile Leu Glu Asn Thr Val Val          35 40 <210> 10 <211> 129 <212> DNA <213> DNA sequence for hMAT-1 <400> 10 gaggaagacc cgtgtgcctg cgagtccctg gtgaaattcc aagccaaagt ggaggggctg 60 ctgcaggccc tgaccaggaa actggaagct gtgagtaagc ggctggccat cctggagaac 120 acagttgtc 129 <210> 11 <211> 45 <212> PRT <213> amino acid sequence for hCOMP <400> 11 Asp Leu Gly Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala   1 5 10 15 Leu Gln Asp Val Arg Glu Leu Leu Arg Gln Gln Val Arg Glu Ile Thr              20 25 30 Phe Leu Lys Asn Thr Val Met Glu Cys Asp Ala Cys Gly          35 40 45 <210> 12 <211> 135 <212> DNA <213> DNA sequence for hCOMP <400> 12 gacctgggcc cgcagatgct tcgggaactg caggaaacca acgcggcgct gcaggacgtg 60 cgggagctgc tgcggcagca ggtcagggag atcacgttcc tgaaaaacac ggtgatggag 120 tgtgacgcgt gcggg 135 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GCN4 <400> 13 ggatccatga aacaacttga agac 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GCN4 <400> 14 gaattcttcg ccaactaatt tctt 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for hMAT1 <400> 15 ggattcgagg aagacccgtg tgcc 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for hMAT1 <400> 16 gaattcgaca actgtgttct ccag 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for hCOMP <400> 17 ggatccgacc tgggcccgca gatg 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for hCOMP <400> 18 gaattccccg cacgcgtcac actc 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for h4-1BBL <400> 19 gaattcagcc cgagactccg cgag 24 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for h4-1BBL <400> 20 gcggccgctt atgggatttc gggggt 26  

Claims (11)

delete A first step of culturing the PBMC (peripheral blood mononuclear cell) isolated from the blood by adding the antigen peptide in a culture medium to form CTL (CD8 + T lymphocyte) specific for the antigen peptide; The antigen peptide-specific CTL was cultured with the first step cultured with a ligand oligomeric matrix protein-human4-1BB (COM-h4-1BB) ligand, which is a pentamer of a peptide consisting of a combination of SEQ ID NO: 11 and SEQ ID NO: 5. And a second step of binding the COMP-h4-1BB ligand, which is a pentamer of the peptide consisting of the binding of SEQ ID NO: 11 and SEQ ID NO: 5; And Comprising a third step of separating only the cells specifically bound to the COMP-h4-1BB ligand which is a pentamer of the peptide consisting of the binding of SEQ ID NO: 11 and SEQ ID NO: 5 from the culture of the second step; A method for isolating antigen specific CD8 + T cells from a blood sample. The method of claim 2, wherein the peptide of the first step is derived from a virus or a cancer antigen. The method of claim 3, wherein the virus-derived peptide is selected from among Cytomegalovirus (CMV) peptide, Epstein-barr Virus (EBV) peptide and human telomerase reverse transcriptase (hTERT) peptide. The method of claim 3, wherein the cancer antigen-derived peptide is WT-1 (Wilm's Tumor-associated gene) peptide. The method of claim 2, wherein the culturing of the first step is incubated for 12 to 16 days. The method of claim 2, wherein the culture medium of the first step is a medium containing autologous plasma or autologous serum, and human IL-2. The method of claim 2, wherein the second step of culturing is characterized in that 1 to 3 hours incubation. delete delete delete

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