KR101756073B1 - Composition for cellular immunotherapy comprising cancer-testis antigen for prevention or treatment of Korean intractable multiple myeloma and preparation method thereof - Google Patents
Composition for cellular immunotherapy comprising cancer-testis antigen for prevention or treatment of Korean intractable multiple myeloma and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a composition for immune therapy or prevention of korean immature multiple myeloma cancer comprising cancer-testis antigens, wherein the dendritic cells loaded with the antigen and the cancer-testis antigen-specific cytotoxic T lymphocyte cells derived therefrom are effective A cytotoxic immune response in cancer immunotherapy can be induced by using a cell immunotherapeutic composition composition for preventing or treating multiple myeloma comprising the compound of the present invention as a vaccine for cancer therapy which is safe and has no side effects It is expected.
Description
The present invention relates to a composition for immune therapy or prevention of Korean intractable multiple myeloma cancer, which comprises a cancer-testis antigen, and more particularly, to a dendritic cell containing the antigen and a cancer-testis antigen-specific cytotoxic T To a cell immunotherapeutic composition for the prevention or treatment of multiple myeloma comprising lymphocyte cells as an active ingredient and a method for producing the same.
Multiple myeloma is a type of blood cancer caused by malignant proliferation of plasma cells. It is the most common disease after malignant lymphoma and acute leukemia in Korea. The incidence rate has increased 30 times in Korea in recent 30 years and it is estimated that there are about 5,000 patients now. It is a disease that can become a social problem due to the rapid increase of such patients.
In the 21st century, the number of patients who received positive treatment results increased due to the introduction of targeted therapies such as thalidomide, bortezomib, and renalidomide in patients with multiple myeloma, but the mean survival rate was still as short as 5 years and most patients died Respectively. In addition, the target therapeutic agent is expensive and difficult to maintain continuous treatment due to serious side effects. Therefore, new therapies with good therapeutic effect and fewer side effects are needed.
In order to overcome the limitations of the conventional methods used for the treatment of cancer, various treatment methods have been studied. Recently, cancer immunotherapy, which shows a cancer-specific therapeutic effect and has no side effects, is attracting attention. This method is known to selectively kill only cancer cells and has little side effect (2nd Novel Immunotherapeutics Summit 2013 Jan 30-Feb 1).
Currently, multiple myeloma is used as a tumor antigen for cancer immunotherapy. However, it is difficult to obtain multiple myeloma cells for treatment from bone marrow in many patients. Therefore, the biggest challenge in the development of cancer immunotherapy using immune cells in multiple myeloma is the development of tumor antigens having specificity for multiple myeloma. It is in urgent need of excavation.
In order to solve the above problems, the present invention provides a composition comprising cancer-testis antigens for prevention and treatment which can safely and safely treat multiple myeloma and increase the tumor-specific immune response without side effects, thereby effectively treating the tumor. And to provide a quantitative method of screening tumor antigen candidate proteins overexpressed from malignant multiple myeloma cells through comparative analysis of expression amounts from cells using the above composition.
The present invention provides a pharmaceutical composition comprising at least one protein selected from the group consisting of cancer-testis antigens CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1, MAGEC2, BAGE1, CTAG2 and SPA17; Dendritic cells loaded with antigenic peptides of the protein; Or cancer-testis antigen-specific cytotoxic T lymphocyte cells as an active ingredient. The present invention also relates to a composition for preventing or treating multiple myeloma.
The present invention also provides a method of treating malignant multiple myeloma cells comprising the steps of: a) preparing malignant multiple myeloma cells; b) identifying purity of malignant multiple myeloma cells; c) quantitatively analyzing malignant multiple myeloma cells harvested at high purity using a primer of cancer-testis antigen to determine overexpression from the cells; And d) preparing the cancer-testis antigen as a recombinant-tumor antigen protein and loading it properly on dendritic cells; To a method for preparing a cell immunotherapeutic composition for preventing or treating multiple myeloma.
The use of a composition comprising a cancer-testicle antigen protein or an antigenic peptide thereof through the present invention is useful as a vaccine for cancer therapy that induces a strong tumor-specific cytotoxic immune response in cancer immunotherapy and is safe and has no side effects It is expected to be able to utilize.
FIG. 1 shows the results of isolation of malignant tumor cells and cell phenotype in bone marrow mononuclear cells of Korean refractory multiple myeloma patients.
FIG. 2 is a result of quantitative analysis of the expression of the cancer-testis antigen SSX2,
FIG. 3 is a result of quantitative analysis of the expression of the cancer-testis antigen SSX42,
4 is a result of quantitative analysis of the expression of the cancer-testis antigen MAGE-A3,
FIG. 5 is a result of quantitative analysis of the expression of the cancer-testis antigen MAGE-C2,
FIG. 6 is a result of quantitative analysis of the expression of cancer-testis antigen CTAG2 (LAGE-1), and FIG. 7 is a graph showing the results of quantitative analysis of cancer-testis antigen-specific CTAG2 This is the result of the target cell recognition reaction using red cell cytotoxic T lymphocyte.
The present invention provides a pharmaceutical composition comprising at least one protein selected from the group consisting of cancer-testis antigens CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1, MAGEC2, BAGE1, CTAG2 and SPA17; Dendritic cells loaded with antigenic peptides of the protein; Or cancer-testis antigen-specific cytotoxic T lymphocyte cells as an active ingredient. The present invention also relates to a composition for preventing or treating multiple myeloma.
Cancer immunotherapy can be classified into tumor-associated antigen (TA) -specific therapy (dendritic cell therapy, peptide vaccine, DNA vaccine, etc.) and TA-nonspecific treatment. Dendritic cells (DCs) are the most potent antigen presenting cells and can induce a tumor antigen-specific immune response to induce a strong anti-cancer immune response. The dendritic cell-based cancer immunotherapy is a method of isolating monocytes from peripheral blood of cancer patients and differentiating and maturing them into cytokines in the test tube and then administering the tumor antigen (TA) to the patient to induce a TA-specific immune response Inducing a strong chemotherapeutic effect can be shown. As mentioned earlier, multiple myeloma is a type of blood cancer caused by malignant proliferation of plasma cells. The most ideal tumor antigen for cancer immunotherapy is not expressed in normal cells, but is expressed specifically in cancer cells, and it is very important to have immunogenecity. When a protein satisfying such conditions is expressed in cancer-testis antigen (cancer-testis antigen).
SSX4 is SEQ ID NO: 3, SSX5 is SEQ ID NO: 4, MAGEA3 is SEQ ID NO: 5, MAGEC1 is SEQ ID NO: 6, MAGEC2 is SEQ ID NO: 7, BAGE1 may have an amino acid sequence of SEQ ID NO: 8, CTAG2 may have an amino acid sequence of SEQ ID NO: 9, and SPA17 may have an amino acid sequence of SEQ ID NO: 10.
The present invention also provides a method of treating malignant multiple myeloma cells comprising the steps of: a) preparing malignant multiple myeloma cells; b) identifying purity of malignant multiple myeloma cells; c) quantitatively analyzing malignant multiple myeloma cells harvested at high purity using a primer of cancer-testis antigen to determine overexpression from the cells; And d) preparing the cancer-testis antigen as a recombinant-tumor antigen protein and loading it properly on dendritic cells; To a method for preparing a cell immunotherapeutic composition for preventing or treating multiple myeloma.
In the present invention, the purity of the malignant multiple myeloma cells is not limited but may be 80% or more, more preferably 90% or more, and more preferably 95% or more, in order to improve the accuracy of the test results.
The primer of step c) of the present invention is characterized in that the gene coding for the CTAG1B protein is a forward primer of SEQ ID NO: 11, a reverse primer of SEQ ID NO: 12; The gene encoding the SSX2 protein comprises the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14; The gene encoding the SSX4 protein comprises a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16; The gene encoding the SSX5 protein comprises the forward primer of SEQ ID NO: 17, the reverse primer of SEQ ID NO: 18; The gene encoding the MAGEA3 protein comprises the forward primer of SEQ ID NO: 19, the reverse primer of SEQ ID NO: 20; The gene encoding the MAGECl protein comprises a forward primer of SEQ ID NO: 21, a reverse primer of SEQ ID NO: 22; The gene encoding the MAGEC2 protein comprises a forward primer of SEQ ID NO: 23, a reverse primer of SEQ ID NO: 24; The gene encoding the BAGE1 protein comprises the forward primer of SEQ ID NO: 25, the reverse primer of SEQ ID NO: 26; The gene encoding the CTAG2 protein comprises a forward primer of SEQ ID NO: 27, a reverse primer of SEQ ID NO: 28; And the SPA17 protein-encoding gene can be amplified using the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30.
In the present invention, quantitative analysis in step c) is not limited, but may be performed by measuring the expression level by a real-time quantitative polymerase chain reaction.
In the present invention, the quantitative analysis is not limited, but the ratio of the expression level to the control group can be measured using beta-actin as a control group.
Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense. In this case, unless there is another specific definition in the technical terms and scientific terms used, it is to be understood that those skilled in the art will understand what is generally understood by those skilled in the art, Descriptions of known functions and configurations that may obscure the gist of the invention are omitted.
[Example 1] Isolation and cellular phenotype of malignant tumor cells in bone marrow mononuclear cells of Korean intractable multiple myeloma patients
MACS (magnetic activated cell separation) method was used for the pure separation of malignant tumor cells. Human pancreatic cell isolation kit II (Miltenyi Biotec) or CD138 microbeads (Miltenyi Biotec) was used as a separation kit for the purity determination of cell phenotype of malignant tumor cells.
These two methods have some differences as follows.
Non-plasma cells were first removed from the bone marrow-mononuclear cells using the kit, positive selection of CD138 positive plasma cells was performed using CD138 microbeads, and separation using CD138 microbeads was performed using the non- And positive selection using CD138 microbeads.
(CD19-PE-Cy5, CD38-PE, CD56-PE-Cy5, and CD138-TITC) of BD cells were used to confirm the cell phenotype of plasma cells positively separated using the above- (5 × 10 4 to 10 5 cells) were treated with 1 μL per 200 μL of PBS (phosphate-buffered saline; Life Technologies) buffer containing 1% serum for 30 min at 4 ° C, And the expression of CD19, CD38, CD56, and CD138 was analyzed using a BD flow cytometer (FACSCalibur, BD).
The standard cell phenotype of malignant multiple myeloma cells is CD19 Low, CD22 negative, CD34 negative, CD56 positive, and CD 138 positive, which corresponds to malignant multiple myeloma cells. The degree of expression thereof is shown in FIG.
[Example 2] Quantitative expression of cancer-testis antigens using real-time qPCR (Real-time qPCR)
High purity total RNA was isolated from the samples obtained from normal human and multiple myeloma patients shown in Table 1 using Rneasy mini kit (Qiagen) and cDNA was synthesized using Primerscript 1st strand cDNA synthesis kit (Takara).
[Table 1]
* Abbreviations are explained in the following description
The SYBR Green PCR Master mix (QUANTI tech) and the 10 cancer-testicular antigens of Table 2 were used for real-time quantitative polymerase chain reaction using each synthesized cDNA as a template. The primers of SEQ ID NOS: 11 to 30 were used for expression.
[Table 2]
* F: Forward (forward), R: Reverse (reverse)
Real-time quantitative PCR was carried out at 95 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 30 seconds. As a control group for the above 10 antigens, the expression level was measured using beta-actin. Quantitative expression levels were determined using the Roger-gene 6 software of RG-3000 instrument and quantitative expression of 10 cancer-testis antigens compared with the control group, beta-actin, using GraphPad Prism 5 software Respectively. The results for some of these antigens are shown in FIGS. 2-6.
As can be seen from the results in FIGS. 2 to 6, the ratio of quantitative expression amounts of the target mRNA and beta-actin was compared with each other. As a result, in the case of multiple myeloma, the ratio of the expression amount in CD138 positive indicating malignant multiple myeloid cells And it is confirmed that it is possible to clearly select the normal person and the patient from this.
[Example 3] Preparation of dendritic cells, preparation of recombinant cancer-testis antigens selected from malignant multiple myeloma cells into dendritic cells and induction of cancer-specific antigen-specific cytotoxic T lymphocytes (CTLs)
The dendritic cells used in the present invention obtained mononuclear cells from peripheral blood of a patient with multiple myeloma obtained by centrifugation using lymphoprep (Ficoll-Hypaque), and then subjected to percoll density gradient or CD14 Monocytes were obtained from positive cells using a magnetic bead.
The resulting monocytes were seeded at a density of 2 × 10 6 cells in a 6-well plate, and 50 ng / mL recombinant human GM-CSF and 20 ng / mL recombinant human IL-4 were seeded at 2-day intervals After 6 days of treatment, the cells were differentiated into immature dendritic cells. Then, the VaxDC cocktail was treated and cultured for 2 days according to the dendritic cell (VaxDC) standard manufacturing protocol (SOP) developed by Park Shelby Co., Ltd. to obtain mature dendritic cells.
From the malignant multiple myeloma cells, 3-5 recombinant cancer-testis antigens selected and prepared were quantitatively analyzed by using the primers of Table 2 in a concentration of 0.5 μg / ml to 2 × 10 5 to 3 × 10 5 cells / (10 μg / ml) for 2 hours at 37 ° C in a CO 2 incubator and co-cultured with CD3 or CD8-positive T lymphocytes to induce cytotoxic T lymphocytes (CTLs).
Three days after co-culture, cytotoxic T lymphocyte cells were proliferated by the addition of 5 ng / mL recombinant human IL-2 and 10 ng / mL IL-7. Then, at intervals of 7 days, the proliferated cytotoxic T lymphocyte cells were stimulated with the antigen-loaded dendritic cells at least two times and co-cultured. After 5 days, the reaction-induced cytotoxic T lymphocyte cells were obtained.
Thus, malignant cells of multiple myeloma cells and multiple myeloma patients overexpressing cancer-testis antigens are co-cultured with cytotoxic T lymphocyte cells as target cells to determine cancer-testis antigen-specific cytotoxicity and its killability Respectively.
[Example 4] Killability of cancer-testis antigen-specific cytotoxic T lymphocyte cells using human interferon-gamma ELISPOT technique
The human interferon-gamma ELISPOT kit (BD) used in this experiment was used in accordance with the manufacturer's protocol (BD TM ELISPOT human IFN-g set, cat no.551849, BD).
Malignant cells of the multiple myeloma cells overexpressing cancer-testis antigen and malignant cells of multiple myeloma were co-cultured in 96-well plates coated with human interferon-gamma antibody for 24 hours using 1:10 or 1:20 ratio as target cells Next, the number of colored human interferon-gamma spots was determined. The number of spots obtained means the number of cancer-testis antigen-specific cytotoxic T lymphocyte cells recognizing cancer-testis antigens expressed in the target cell.
The degree of secretion of interferon gamma was confirmed by specifically recognizing the target cell by cytotoxic T lymphocyte cells specifically prepared from the dendritic cells in which the cancer-testis antigens were selectively produced.
The experimental results are as follows.
The K562 cell line is a cell line that is dependent on NK cells and the RPMI8226 and U266 cell lines are multiple myeloma cell lines (n = 2) overexpressing cancer-testis antigens and P3, P4, P5 and P6 are overexpressing cancer- It is a multiple myeloma cell of multiple myeloma (n = 4).
Un-CTL is a cytotoxic T lymphocyte cell induced by dendritic cells not loaded with the above-described cancer-testis antigen, and an antigen-nonspecific reaction can be confirmed from the blue graph of FIG.
SSX2-, SSX4-, MAGEA3- and MAGEC2-CTL are cancer-testis antigen-specific cytotoxic T lymphocyte cells (red graph in FIG. 7) induced by dendritic cells loaded with cancer-testis antigens.
In the graph of FIG. 7, the interval of the spots indicates the intensity of the cancer-testis antigen-specific cytotoxic T lymphocyte reaction, and the larger the distance between the blue and red graphs, the stronger the antigen-specific reaction intensity .
As can be seen from the results of FIG. 7, in the case of cytotoxic T lymphocyte cells induced by cancer-testis antigens, a spot that secretes interferon gamma in response to a target cell overexpressing cancer-testis antigen was specifically and strongly produced, Since the reaction is specifically inhibited by a monoclonal antibody of MHC (major histocompatibility complex) molecule I, it has been confirmed that it is an antigen-specific reaction mediated by MHC molecule I. Thus, the present invention has been completed.
PB-peripheral blood mononuclear cells, BB cells, BM-bone marrow mononuclear cells, IgA, -immunoglobulin A, IgG-immunoglobulin G, lamda-lamda light chain, kappa-kappa light chain , CD138 ?? -CD138 negative multiple myeloma cells, CD138 + -CD138 multiple myeloma cells, MACS-magnectic activated cell separation.
<110> University Industry Liaison Office of Chonnam National University VaxCell-Bio <120> Composition for cellular immunotherapy comprising cancer-testis antigen for prevention or treatment of Korean intractable multiple myeloma and preparation method thereof <130> P15040960464 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> CTAG1B (CT6.1) <400> 1 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly Pro Gly Gly Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110 Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175 Gly Gln Arg Arg 180 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> SSX2 (CT5.2a) <400> 2 Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala Gln 1 5 10 15 Ile Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30 Ser Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr 35 40 45 Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys 50 55 60 Ala Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe Gln 65 70 75 80 Gly Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu Arg 85 90 95 Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met 100 105 110 Pro Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro Glu 115 120 125 Ala Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly Lys 130 135 140 Pro Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Asn Arg Glu Ala 145 150 155 160 Gln Glu Lys Glu Glu Arg Arg Gly Thr Ala His Arg Trp Ser Ser Gln 165 170 175 Asn Thr His Asn Ile Gly Pro Lys Arg Gly Glu His Ala Trp Thr His 180 185 190 Arg Leu Arg Glu Arg Lys Gln Leu Val Ile Tyr Glu Glu Ile Ser Asp 195 200 205 Pro Glu Glu Asp Asp Glu 210 <210> 3 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> SSX4 (CT5.4) <400> 3 Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Arg Asp Asp Ala Gln 1 5 10 15 Ile Ser Glu Lys Leu Arg Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30 Ser Lys Lys Glu Trp Glu Lys Met Lys Ser Ser Glu Lys Ile Val Tyr 35 40 45 Val Tyr Met Lys Leu Asn Tyr Glu Val Met Thr Lys Leu Gly Phe Lys 50 55 60 Val Thr Leu Pro Pro Phe Met Arg Ser Lys Arg Ala Ala Asp Phe His 65 70 75 80 Gly Asn Asp Phe Gly Asn Asp Arg Asn His Arg Asn Gln Val Glu Arg 85 90 95 Pro Gln Met Thr Phe Gly Ser Leu Gln Arg Ile Phe Pro Lys Ile Met 100 105 110 Pro Lys Lys Pro Ala Glu Glu Glu Asn Gly Leu Lys Glu Val Pro Glu 115 120 125 Ala Ser Gly Pro Gln Asn Asp Gly Lys Gln Leu Cys Pro Pro Gly Asn 130 135 140 Pro Ser Thr Leu Glu Lys Ile Asn Lys Thr Ser Gly Pro Lys Arg Gly 145 150 155 160 Lys His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Val 165 170 175 Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu 180 185 <210> 4 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> SSX5 (CT5.5) <400> 4 Met Asn Gly Asp Asp Ala Phe Val Arg Arg Pro Arg Val Gly Ser Gln 1 5 10 15 Ile Pro Glu Lys Met Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30 Ser Glu Lys Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Ile Tyr 35 40 45 Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys 50 55 60 Ala Thr Leu Pro Pro Phe Met Arg Asn Lys Arg Val Ala Asp Phe Gln 65 70 75 80 Gly Asn Asp Phe Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu His 85 90 95 Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Phe Pro Lys Ile Thr 100 105 110 Pro Glu Lys Pro Ala Glu Glu Gly Asn Asp Ser Lys Gly Val Pro Glu 115 120 125 Ala Ser Gly Pro Gln Asn Asn Gly Lys Gln Leu Arg Pro Ser Gly Lys 130 135 140 Leu Asn Thr Ser Glu Lys Val Asn Lys Thr Ser Gly Pro Lys Arg Gly 145 150 155 160 Lys His Ala Trp Thr His Arg Val Arg Glu Arg Lys Gln Leu Val Ile 165 170 175 Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu 180 185 <210> 5 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> MAGEA3 (CT1.3) <400> 5 Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5 10 15 Gly Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30 Thr Glu Glu Glu Glu Ala Ala Ser Ser Ser Thr Leu Val Glu Val 35 40 45 Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60 Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp 65 70 75 80 Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95 Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Leu Ser Arg Lys 100 105 110 Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125 Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135 140 Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu 145 150 155 160 Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175 Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190 Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205 Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220 Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly 225 230 235 240 Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 245 250 255 Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260 265 270 Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285 His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300 His Glu Trp Val Leu Arg Glu Gly Glu Glu 305 310 <210> 6 <211> 1142 <212> PRT <213> Artificial Sequence <220> <223> MAGEC1 (CT7.1) <400> 6 Met Gly Asp Lys Asp Met Pro Thr Ala Gly Met Pro Ser Leu Leu Gln 1 5 10 15 Ser Ser Glu Ser Pro Gln Ser Cys Pro Glu Gly Glu Asp Ser Gln 20 25 30 Ser Pro Leu Gln Ile Pro Gln Ser Ser Pro Glu Ser Asp Asp Thr Leu 35 40 45 Tyr Pro Leu Gln Ser Pro Gln Ser Ser Ser Glu Gly Glu Asp Ser Ser 50 55 60 Asp Pro Leu Gln Arg Pro Pro Glu Gly Lys Asp Ser Gln Ser Pro Leu 65 70 75 80 Gln Ile Pro Gln Ser Ser Pro Glu Gly Asp Asp Thr Gln Ser Pro Leu 85 90 95 Gln Asn Ser Gln Ser Ser Pro Glu Gly Lys Asp Ser Leu Ser Pro Leu 100 105 110 Glu Ile Ser Gln Ser Pro Pro Glu Gly Glu Asp Val Glu Ser Ser Leu 115 120 125 Gln Asn Pro Ala Ser Ser Phe Phe Ser Ser Ala Leu Ser Ile Phe 130 135 140 Gln Ser Ser Pro Glu Ser Thr Gln Ser Pro Phe Glu Gly Phe Pro Gln 145 150 155 160 Ser Val Leu Gln Ile Pro Val Ser Ala Ser Ser Ser Thr Leu Val 165 170 175 Ser Ile Phe Gln Ser Ser Pro Glu Ser Thr Gln Ser Pro Phe Glu Gly 180 185 190 Phe Pro Gln Ser Pro Leu Gln Ile Pro Val Ser Ser Arg Ser Ser Ser Ser 195 200 205 Thr Leu Leu Ser Ile Phe Gln Ser Ser Pro Glu Arg Thr Gln Ser Thr 210 215 220 Phe Glu Gly Phe Ala Gln Ser Pro Leu Gln Ile Pro Val Ser Ser Ser 225 230 235 240 Ser Ser Thr Leu Leu Ser Leu Phe Gln Ser Phe Ser Glu Arg Thr 245 250 255 Gln Ser Thr Phe Glu Gly Phe Ala Gln Ser Ser Leu Gln Ile Pro Val 260 265 270 Ser Pro Ser Phe Ser Ser Thr Leu Val Ser Leu Phe Gln Ser Ser Pro 275 280 285 Glu Arg Thr Gln Ser Thr Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln 290 295 300 Ile Pro Val Ser Ser Ser Ser Ser Thr Leu Leu Ser Leu Phe Gln 305 310 315 320 Ser Ser Pro Glu Arg Thr His Ser Thr Phe Glu Gly Phe Pro Gln Ser 325 330 335 Leu Leu Gln Ile Pro Met Thr Ser Ser Phe Ser Ser Thr Leu Leu Ser 340 345 350 Ile Phe Gln Ser Ser Pro Glu Ser Ala Gln Ser Thr Phe Glu Gly Phe 355 360 365 Pro Gln Ser Pro Leu Gln Ile Pro Gly Ser Pro Ser Phe Ser Ser Thr 370 375 380 Leu Leu Ser Leu Phe Gln Ser Ser Pro Glu Arg Thr His Ser Thr Phe 385 390 395 400 Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile Pro Met Thr Ser Ser Phe 405 410 415 Ser Ser Thr Leu Leu Ser Ile Leu Gln Ser Ser Pro Glu Ser Ala Gln 420 425 430 Ser Ala Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile Pro Val Ser 435 440 445 Ser Ser Phe Ser Tyr Thr Leu Leu Ser Leu Phe Gln Ser Ser Ser Glu 450 455 460 Arg Thr His Ser Thr Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile 465 470 475 480 Pro Val Ser Ser Ser Ser Ser Ser Thr Leu Leu Ser Leu Phe Gln 485 490 495 Ser Ser Pro Glu Cys Thr Gln Ser Thr Phe Glu Gly Phe Pro Gln Ser 500 505 510 Pro Leu Gln Ile Pro Gln Ser Pro Pro Glu Gly Glu Asn Thr His Ser 515 520 525 Pro Leu Gln Ile Val Pro Ser Leu Pro Glu Trp Glu Asp Ser Leu Ser 530 535 540 Pro His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Leu Ser 545 550 555 560 Pro His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Leu Ser 565 570 575 Pro His Tyr Phe Pro Gln Ser Pro Gln Gly Glu Asp Ser Leu Ser Pro 580 585 590 His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Ser Ser Pro 595 600 605 Leu Tyr Phe Pro Gln Ser Pro Leu Gln Gly Glu Glu Phe Gln Ser Ser 610 615 620 Leu Gln Ser Ser Val Ser Ser C Ser Ser Ser Ser Ser Ser Ser Le 625 630 635 640 Pro Gln Ser Phe Pro Glu Ser Ser Gln Ser Pro Pro Glu Gly Pro Val 645 650 655 Gln Ser Pro Leu His Ser Pro Gln Ser Pro Pro Glu Gly Met His Ser 660 665 670 Gln Ser Pro Leu Gln Ser Pro Glu Ser Ala Pro Glu Gly Glu Asp Ser 675 680 685 Leu Ser Pro Leu Gln Ile Pro Gln Ser Pro Leu Glu Gly Glu Asp Ser 690 695 700 Leu Ser Ser Leu His Phe Pro Gln Ser Pro Pro Glu Trp Glu Asp Ser 705 710 715 720 Leu Ser Pro Leu His Phe Pro Gln Phe Pro Pro Gln Gly Glu Asp Phe 725 730 735 Gln Ser Ser Leu Gln Ser Ser Val Ser Ser Ser Ser Thr Ser 740 745 750 Leu Ser Leu Pro Gln Ser Phe Pro Glu Ser Pro Gln Ser Pro Pro Glu 755 760 765 Gly Pro Ala Gln Ser Pro Leu Gln Arg Pro Val Ser Ser Phe Phe Ser 770 775 780 Tyr Thr Leu Ala Ser Leu Leu Gln Ser Ser His Glu Ser Pro Gln Ser 785 790 795 800 Pro Pro Glu Gly Pro Ala Gln Ser Pro Leu Gln Ser Pro Val Ser Ser 805 810 815 Phe Pro Ser Ser Thr Ser Ser Leu Ser Gln Ser Ser Ser Val Ser 820 825 830 Ser Phe Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 835 840 845 Ser Pro Leu Gln Ser Ser Val Ser Ser Thr Ser Leu 850 855 860 Ser Pro Phe Ser Glu Glu Ser Ser Ser Pro Val Asp Glu Tyr Thr Ser 865 870 875 880 Ser Ser Asp Thr Leu Leu Glu Ser Asp Ser Leu Thr Asp Ser Glu Ser 885 890 895 Leu Ile Glu Ser Glu Pro Leu Phe Thr Tyr Thr Leu Asp Glu Lys Val 900 905 910 Asp Glu Leu Ala Arg Phe Leu Leu Leu Lys Tyr Gln Val Lys Gln Pro 915 920 925 Ile Thr Lys Ala Glu Met Leu Thr Asn Val Ile Ser Arg Tyr Thr Gly 930 935 940 Tyr Phe Pro Val Ile Phe Arg Lys Ala Arg Glu Phe Ile Glu Ile Leu 945 950 955 960 Phe Gly Ile Ser Leu Arg Glu Val Asp Pro Asp Ser Ser Val Val Phe 965 970 975 Val Asn Thr Leu Asp Leu Thr Ser Glu Gly Cys Leu Ser Asp Glu Gln 980 985 990 Gly Met Ser Gln Asn Arg Leu Leu Ile Leu Ile Leu Ser Ile Ile Phe 995 1000 1005 Ile Lys Gly Thr Tyr Ala Ser Glu Glu Val Ile Trp Asp Val Leu Ser 1010 1015 1020 Gly Ile Gly Val Arg Ala Gly Arg Glu His Phe Ala Phe Gly Glu Pro 1025 1030 1035 1040 Arg Glu Leu Leu Thr Lys Val Trp Val Gln Glu His Tyr Leu Glu Tyr 1045 1050 1055 Arg Glu Val Pro Asn Ser Ser Pro Pro Arg Tyr Glu Phe Leu Trp Gly 1060 1065 1070 Pro Arg Ala His Ser Glu Val Ile Lys Arg Lys Val Val Glu Phe Leu 1075 1080 1085 Ala Met Leu Lys Asn Thr Val Pro Ile Thr Phe Pro Ser Ser Tyr Lys 1090 1095 1100 Asp Ala Leu Lys Asp Val Glu Glu Arg Ala Gln Ala Ile Ile Asp Thr 1105 1110 1115 1120 Thr Asp Asp Ser Thr Ala Thr Glu Ser Ala Ser Ser Ser Val Met Ser 1125 1130 1135 Pro Ser Phe Ser Ser Glu 1140 <210> 7 <211> 373 <212> PRT <213> Artificial Sequence <220> <223> MAGEC2 (CT10) <400> 7 Met Pro Pro Val Gly Val Pro Phe Arg Asn Val Asp Asn Asp Ser 1 5 10 15 Pro Thr Ser Val Glu Leu Glu Asp Trp Val Asp Ala Gln His Pro Thr 20 25 30 Asp Glu Glu Glu Glu Glu Ala Ser Ser Ala Ser Ser Thr Leu Tyr Leu 35 40 45 Val Phe Ser Ser Ser Ser Phe Ser Ser Ser Ser Leu Ile Leu Gly 50 55 60 Gly Pro Glu Glu Glu Glu Val Pro Ser Gly Val Ile Pro Asn Leu Thr 65 70 75 80 Glu Ser Ile Pro Ser Ser Pro Pro Gln Gly Pro Pro Gln Gly Pro Ser 85 90 95 Gln Ser Pro Leu Ser Ser Cys Cys Ser Ser Phe Ser Trp Ser Ser Phe 100 105 110 Ser Glu Ser Ser Ser Gln Lys Gly Glu Asp Thr Gly Thr Cys Gln 115 120 125 Gly Leu Pro Asp Ser Glu Ser Ser Phe Thr Tyr Thr Leu Asp Glu Lys 130 135 140 Val Ala Glu Leu Val Glu Phe Leu Leu Leu Lys Tyr Glu Ala Glu Glu 145 150 155 160 Pro Val Thr Glu Ala Glu Met Leu Met Ile Val Ile Lys Tyr Lys Asp 165 170 175 Tyr Phe Pro Val Ile Leu Lys Arg Ala Arg Glu Phe Met Glu Leu Leu 180 185 190 Phe Gly Leu Ala Leu Ile Glu Val Gly Pro Asp His Phe Cys Val Phe 195 200 205 Ala Asn Thr Val Gly Leu Thr Asp Glu Gly Ser Asp Asp Glu Gly Met 210 215 220 Pro Glu Asn Ser Leu Leu Ile Ile Ile Leu Ser Val Ile Phe Ile Lys 225 230 235 240 Gly Asn Cys Ala Ser Glu Glu Val Ile Trp Glu Val Leu Asn Ala Val 245 250 255 Gly Val Tyr Ala Gly Arg Glu His Phe Val Tyr Gly Glu Pro Arg Glu 260 265 270 Leu Leu Thr Lys Val Trp Val Gln Gly His Tyr Leu Glu Tyr Arg Glu 275 280 285 Val Pro His Ser Ser Pro Pro Tyr Tyr Glu Phe Leu Trp Gly Pro Arg 290 295 300 Ala His Ser Glu Ser Ile Lys Lys Lys Val Leu Glu Phe Leu Ala Lys 305 310 315 320 Leu Asn Asn Thr Val Ser Ser Phe Pro Ser Trp Tyr Lys Asp Ala 325 330 335 Leu Lys Asp Val Glu Glu Arg Val Gln Ala Thr Ile Asp Thr Ala Asp 340 345 350 Asp Ala Thr Val Met Ala Ser Glu Ser Leu Ser Val Met Ser Ser Asn 355 360 365 Val Ser Phe Ser Glu 370 <210> 8 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> BAGE1 (CT2.1) <400> 8 Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln 1 5 10 15 Ala Arg Leu Met Lys Glu Glu Ser Pro Val Val Ser Trp Arg Leu Glu 20 25 30 Pro Glu Asp Gly Thr Ala Leu Cys Phe Ile Phe 35 40 <210> 9 <211> 180 <212> PRT <213> Artificial Sequence <220> ≪ 223 > CTAG2 (CT6.2a: LAGE-1) <400> 9 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly Pro Gly Gly Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala 65 70 75 80 Arg Arg Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr Met Pro Phe 85 90 95 Ser Ser Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu Ser Arg Asp 100 105 110 Ala Ala Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp Phe Thr Val 115 120 125 Ser Gly Asn Leu Leu Phe Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Ala Pro Ser 165 170 175 Gly Gln Arg Arg 180 <210> 10 <211> 151 <212> PRT <213> Artificial Sequence <220> ≪ 223 > SPA17 (CT22) <400> 10 Met Ser Ile Pro Phe Ser Asn Thr His Tyr Arg Ile Pro Gln Gly Phe 1 5 10 15 Gly Asn Leu Leu Glu Gly Leu Thr Arg Glu Ile Leu Arg Glu Gln Pro 20 25 30 Asp Asn Ile Pro Ala Phe Ala Ala Tyr Phe Glu Ser Leu Leu Glu 35 40 45 Lys Arg Glu Lys Thr Asn Phe Asp Pro Ala Glu Trp Gly Ser Lys Val 50 55 60 Glu Asp Arg Phe Tyr Asn Asn His Ala Phe Glu Glu Gln Glu Pro Pro 65 70 75 80 Glu Lys Ser Asp Pro Lys Gln Glu Glu Ser Gln Ile Ser Gly Lys Glu 85 90 95 Glu Glu Thr Ser Val Thr Ile Leu Asp Ser Ser Glu Glu Asp Lys Glu 100 105 110 Lys Glu Glu Val Ala Ala Val Lys Ile Gln Ala Ala Phe Arg Gly His 115 120 125 Ile Ala Arg Glu Glu Ala Lys Lys Met Lys Thr Asn Ser Leu Gln Asn 130 135 140 Glu Glu Lys Glu Glu Asn Lys 145 150 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CTAG1B (CT6.1) Forward <400> 11 ggccctgacc ttctctctg 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CTAG1B (CT6.1) Reverse <400> 12 cctgggccat caggaatg 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX2 (CT5.2a) Forward <400> 13 agcatctgcc atctgctttt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX2 (CT5.2a) Reverse <400> 14 gaggggagta catgctgacc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX4 (CT5.4) Forward <400> 15 cttgcagtgt gccattctgt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX4 (CT5.4) Reverse <400> 16 ggggagagag gagggtagtg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX5 (CT5.5) Forward <400> 17 ttcccagatg tgaccactga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSX5 (CT5.5) Reverse <400> 18 gacacatggg gaaaagcagt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEA3 (CT1.3) Forward <400> 19 ctccagcaac caagaagagg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEA3 (CT1.3) Reverse <400> 20 cagcatttct gcctttgtga 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEC1 (CT7.1) Forward <400> 21 aggcagagat gctgacgaat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEC1 (CT7.1) Reverse <400> 22 ccctgctcat cactcagaca 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEC2 (CT10) Forward <400> 23 tgcctctgag gaggtcatct 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MAGEC2 (CT10) Reverse <400> 24 gtccctgcac ccaaacttta 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAGE1 (CT2.1) Forward <400> 25 ccaatttagg gtctccggta 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAGE1 (CT2.1) Reverse <400> 26 caccatccag agtgagacga 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CTAG2 (CT6.2a: LAGE-1) Forward <400> 27 agctttccct gttgatgtgg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> ≪ 223 > CTAG2 (CT6.2a: LAGE-1) Reverse <400> 28 ctcctccagc gacaaacaat 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPA17 (CT22) Forward <400> 29 ccattgaaac atgccacttg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPA17 (CT22) Reverse <400> 30 tgggggatgt gatagagagg 20
Claims (6)
b) confirming the purity of the malignant multiple myeloma cells;
c) expression of SSX2 protein having the amino acid sequence of SEQ ID NO: 2, which is a cancer-testis antigen, from a malignant multiple myeloma cell harvested at a high purity by real-time quantitative polymerase chain reaction using a primer (Beta-actin) as a control group through quantitative analysis to determine the overexpression from the cells by measuring the ratio of the expression amount to the control group; And
d) preparing the cancer-testis antigen as a recombinant-tumor antigen protein and loading it on dendritic cells;
Wherein the method comprises the step of administering to a patient a therapeutically effective amount of a cell-mediated immunotherapeutic composition for preventing or treating multiple myeloma.
The primer of step c)
The gene encoding the SSX2 protein comprises the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14;
Is used.
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| KR1020150140390A KR101756073B1 (en) | 2015-10-06 | 2015-10-06 | Composition for cellular immunotherapy comprising cancer-testis antigen for prevention or treatment of Korean intractable multiple myeloma and preparation method thereof |
| PCT/KR2016/011178 WO2017061784A1 (en) | 2015-10-06 | 2016-10-06 | Cellular immunotherapeutic agent composition for preventing or treating korean intractable multiple myeloma, containing cancer-testis antigen, and preparation method therefor |
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| KR1020150140390A KR101756073B1 (en) | 2015-10-06 | 2015-10-06 | Composition for cellular immunotherapy comprising cancer-testis antigen for prevention or treatment of Korean intractable multiple myeloma and preparation method thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Blood, Vol. 100, pages 961-965 (2002)* |
| Blood, Vol. 109, pages 1103-1112 (2007)* |
| Haematologica, Vol. 96, pages 1662-1669(2011)* |
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