WO2006040920A1 - Procédé pour évaluer la capacité d’induction ctl et procédé de criblage utilisant celui-ci - Google Patents

Procédé pour évaluer la capacité d’induction ctl et procédé de criblage utilisant celui-ci Download PDF

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Publication number
WO2006040920A1
WO2006040920A1 PCT/JP2005/017724 JP2005017724W WO2006040920A1 WO 2006040920 A1 WO2006040920 A1 WO 2006040920A1 JP 2005017724 W JP2005017724 W JP 2005017724W WO 2006040920 A1 WO2006040920 A1 WO 2006040920A1
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Prior art keywords
protein
antigen
cell
ctl
presenting
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PCT/JP2005/017724
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English (en)
Japanese (ja)
Inventor
Kazuhiro Kakimi
Mie Nieda
Toshimasa Tadaki
Shigemi Sasawatari
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Medinet Co., Ltd.
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Priority to JP2006540866A priority Critical patent/JPWO2006040920A1/ja
Publication of WO2006040920A1 publication Critical patent/WO2006040920A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis

Definitions

  • the present invention relates to a method for evaluating CTL inducibility and a screening method using the same.
  • Immunity is a series of biological defense systems and other recognition systems that work against what is recognized as a foreign substance (antigen) in a living body. This system protects organisms from various diseases.
  • immune cell therapy that artificially enhances cellular immunity and treats various diseases (particularly cancer and viral infections) has attracted attention.
  • the advantage of immune cell therapy is that it only requires a mild fever, even if it has very few side effects.
  • CTL therapy cytotoxic T cell therapy
  • CTL therapy is said to be particularly effective.
  • Cytotoxic T Lymphocytes (hereinafter also referred to as CTL) play a central role in biological defense reactions that recognize and eliminate tumors (cancer cells) and viruses.
  • CTL therapy is a treatment / prevention method that induces CTLs specific to these antigens for the purpose of treatment and prevention of cancer and infectious diseases, and plays a central role in the immune response to cancer and viruses. It is a treatment / prevention method aimed at inducing CTL and eliminating cancer cells and virus-infected cells.
  • CTL recognizes a part of a protein that is specifically expressed on or in the surface of cancer cells or virus-infected cells and is called an antigen epitope peptide.
  • Antigen epitope peptides usually also have 9 to: L 1 amino acid residue strength.
  • This antigen epitope peptide binds to major histocompatibility antigen complex (MHC) class I and is presented on antigen-presenting cells.
  • MHC major histocompatibility antigen complex
  • Re The lymphocyte recognizes the presented antigen epitope peptide and becomes a CTL that specifically attacks cancer cells or virus-infected cells having the same antigen epitope peptide (for example, see Non-Patent Document 1). ).
  • a tumor-specific CTL cell line must be established.
  • DC must be induced every time as an antigen-presenting cell and cultured.
  • Non-patent literature 1 EMS Boon et al, Tumor antigens recognized by T cells "Immunology today 267—268 Vol. 18, 1997
  • Non-Patent Document 2 Joachim L. Schultzeand et al, "From cancer genomics to cancer immunotherapy: toward second-generation tumor antigens" Trends in Immunology Vol. 22 No. 9: 516-523, 2001 Disclosure of the Invention
  • the present invention has been made in view of the above circumstances, and has a specific CTL inducing ability that enables rapid and simple screening of an antigenic epitope peptide without using DC as an antigen-presenting cell.
  • the purpose is to provide an evaluation method.
  • a T cell costimulatory factor and an MHC class I molecule are expressed, and all or part of the protein expressed intracellularly can be presented as an antigen on the MHC class I molecule. This is an evaluation method using cells derived from a cell line. The invention's effect
  • the present inventors have heretofore been used as antigen-presenting cells for screening methods using CTL specifically induced by antigen epitope peptides as an index.
  • DC we intensively researched the idea of using an established cell line that has excellent proliferative ability and does not require differentiation induction.
  • the antigen-presenting cell is derived from a cell line that expresses or has been modified to express a T cell costimulatory factor and an MHC class I molecule, it encodes a protein containing an antigen epitope peptide.
  • the gene to be expressed is introduced into the antigen-presenting cell so that it can be expressed, the antigen epitope peptide is presented as an antigen on the MHC class I molecule and a specific CTL can be induced. Furthermore, the present inventors have shown that the efficiency of gene introduction into the antigen-presenting cells is superior to that of conventional DC, and therefore the specific CTL inducing ability of the introduced protein can be improved by using the antigen-presenting cells. It has been found that screening can be performed more quickly and simply than conventional methods, and by using this evaluation method, screening of antigen epitope peptides can be performed quickly and simply.
  • the "specific CTL inducing ability” means a protein or peptide power that is specific to the above-mentioned epitope peptide when part or all of the protein or peptide strength is presented as an epitope peptide. It means the ability to activate CTL and promote proliferation.
  • “established cell line” means a cell line capable of producing clonal cells having proliferative ability and exhibiting the same properties and characteristics.
  • the antigen-presenting cell of the present invention since the antigen-presenting cell of the present invention has excellent gene transfer efficiency, the necessary preparatory work can be efficiently performed in a shorter time. Therefore, according to the present invention, it is possible to provide a quick and simple method for evaluating CTL inducibility. Further, the gene introduction in the evaluation method of the present invention can be a rapid and simpler evaluation method of the present invention, which may be a transient introduction.
  • the evaluation method of the present invention even if a gene encoding the whole protein is introduced regardless of the size of the protein, it is specific to the antigen epitope peptide contained in the protein. Since the ability to induce CTL can be evaluated, a method for screening a protein containing an antigenic epitope peptide that induces a specific CTL is possible. In addition, if the gene to be introduced is a gene encoding a part of the protein and the site or length of the protein is changed, screening of antigen epitope peptides can be performed by the evaluation method of the present invention. Is possible.
  • a protein or antigenic epitope peptide that specifically induces CTL without establishing an antigen-specific CTL cell line or synthesizing a peptide of an antigenic epitope candidate. This screening can be performed quickly and easily.
  • FIG. 1 is an example of a flow cytometer analysis diagram of the MDA-CD80 cell line.
  • FIG. 2 is a diagram showing an example of RT-PCR results for the MDA-CD80 cell line.
  • FIG. 3A is an example of an example in which pTracer was introduced into an MDA-CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducibility was measured by FACS. .
  • Figure 3B shows an example of MDA-CD80 cell line introduced with pTracer-BMLF 1 for antigen presentation, co-cultured with lymphocytes, and specific CTL inducibility measured by FACS. It is.
  • FIG. 3C shows an example in which pTracer—Ub—BMLF 1 was introduced into an MDA CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducing ability was measured by FACS. It is an example figure.
  • FIG. 4A is an example of introduction of pTracer into an MDA-CD80 cell line for antigen presentation, co-culture with lymphocytes, and measurement of specific CTL inducing ability by FACS. .
  • FIG. 4B shows an example in which pTracer-Marti was introduced into an MDA-CD80 cell line for antigen presentation, co-cultured with lymphocytes, and specific CTL inducing ability was measured by FACS. is there.
  • FIG. 4C shows the antigen presentation by introducing pTracer-Ub-Mart 1 into the MDA-CD80 cell line, co-cultured with lymphocytes, and the specific CTL inducing ability was measured by FACS.
  • Fig. 5A shows the introduction of pTracer into an MDA-CD80 cell line for antigen presentation, co-culture with lymphocytes, and the ability to induce specific CTLs using INF- ⁇ as an index. It is a figure of an example measured by.
  • Fig. 5B shows the introduction of pTracer—Ub—BMLF 1 into the MDA CD80 cell line, antigen presentation, co-culture with lymphocytes, and specific CTL induction using INF-y as one of the indicators. It is a figure of an example which measured conductivity with FACS.
  • the T cell costimulatory factor is preferably CD80.
  • a gene encoding ubiquitin in addition to the protein, it is preferable to introduce a gene encoding ubiquitin so that it can be expressed. More preferably, the gene encoding a fusion protein of the protein and ubiquitin is used. Is introduced in an expressible manner.
  • the gene transfer may be a transient gene transfer.
  • the protein may be derived from a cancer cell or a virus.
  • the antigen-presenting cell is preferably adherent, and is preferably a cell derived from a tumor cell-derived cell line.
  • the tumor cell-derived cell line is preferably a breast cancer-derived cell line MDA-MB-231! /.
  • the screening method of the present invention is a protein screening method capable of specifically inducing CTL (hereinafter also referred to as the antigen protein screening method of the present invention).
  • Evaluate specific CTL inducibility of test protein A screening method comprising a step of identifying a protein having a typical ability to induce CTL.
  • the screening method of the present invention is a method for screening an antigenic epitope peptide in a protein capable of specifically inducing CTL (hereinafter, the screening method for an antigenic epitope peptide of the present invention). It is also a screening method comprising at least one of the following steps (A) and (B).
  • a polynucleotide encoding a partial peptide of the protein is introduced into the antigen-presenting cell so that it can be expressed.
  • the test protein is preferably a protein identified by the antigen protein screening method of the present invention.
  • the partial peptide may be a peptide having 9 to L 1 residues.
  • the antigen-presenting cell of the present invention is an antigen-presenting cell used in the evaluation method of the present invention, and expresses a T cell costimulatory factor and an MHC class I molecule, and is expressed in the cell.
  • Antigen-presenting cells derived from an established cell line capable of presenting all or part of the antigen on the MHC class I molecule.
  • the antigen-presenting cell of the present invention is the antigen-presenting cell used in the screening method for the antigen protein of the present invention as another aspect, and as another aspect, the antigen-epitope peptide of the present invention is screened.
  • the antigen-presenting cell used in the method is screened.
  • the kit of the present invention is an evaluation kit used in the evaluation method of the present invention, and includes the antigen-presenting cell of the present invention.
  • the kit of the present invention is a screening kit used in the method for screening an antigen protein of the present invention, and includes the antigen-presenting cell of the present invention.
  • the kit of the present invention is a screening kit used in the screening method for an antigenic epitope peptide of the present invention as yet another embodiment.
  • the production method of the present invention is a method for producing an antigenic epitope peptide having a specific CTL inducing ability, and the method for screening an antigenic epitope peptide of the present invention allows the antigen epitope of the protein to be produced.
  • a production method comprising the step of identifying a peptide.
  • the production method of the present invention preferably further includes at least one of a step of separating the antigen epitope peptide identified with the protein strength and a step of synthesizing the identified antigen epitope peptide.
  • the evaluation method of the present invention uses the antigen-presenting cell of the present invention which is a cell line that expresses a T cell costimulatory factor and an MHC class I molecule, and the test protein is applied to the antigen-presenting cell.
  • One of the technical features is to introduce the encoded gene and present the antigen.
  • test protein In the antigen-presenting cell, transcription and translation of the introduced gene is performed, and a test protein is synthesized.
  • the synthesized test protein is decomposed into several fragments by intracellular proteolytic enzymes (hereinafter also referred to as proteasomes).
  • proteasomes intracellular proteolytic enzymes
  • T cell receptor present on the surface of T lymphocytes recognizes a complex of MHC class I molecules and epitope peptides presented on the surface of antigen-presenting cells. To do. T lymphocytes with TCR binding to epitopes are activated to CTL.
  • an antigen that can bind to an MHC class I molecule in a test protein and is recognized by TCR is confirmed by confirming a specific CTL inducing ability. It is possible to easily confirm the presence of the pitope peptide.
  • An example of the evaluation method of the present invention will be specifically described below.
  • a vector incorporating a gene encoding a test protein for evaluating specific CTL inducing ability is prepared and introduced into the antigen-presenting cell of the present invention.
  • the test protein is not particularly limited and refers to a protein in which an amino acid has a peptide bond and has a linear force S, and may be a simple protein that has only an amino acid force. It may be a complex protein (eg, glycoprotein, nucleoprotein, lipoprotein, heme protein, metal protein, etc.). Further, the number of amino acid residues is not particularly limited, and may be, for example, an oligopeptide or a polypeptide.
  • the method for preparing the vector into which the gene is incorporated is not particularly limited, and a conventionally known method can be applied. For example, after obtaining cDNA (complementary DNA; complementary deoxyribonucleic acid) of a gene to be expressed. For example, a method of amplifying cDNA by PCR method (Polymerase Chain Reaction method; polymerase chain reaction method) and incorporating it into an appropriate expression vector can be mentioned.
  • the expression vector is not particularly limited, and examples thereof include a plasmid vector and a virus vector.
  • the size of the gene is not particularly limited. For example, even when a gene encoding the full length of the test protein is used, the procedure is very simple because it undergoes proteolysis in the cell and presents the epitope site as an antigen. Further, the gene may be a polynucleotide encoding a part of the test protein.
  • test protein for example, a protein derived from cancer cells can be used.
  • target cancer cells include liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, uterine cancer, brain tumor and the like.
  • an antigen epitope peptide is identified and the peptide is synthesized, and the synthesized peptide is administered to a cancer patient as a peptide vaccine.
  • DC rod cells
  • test protein for example, a virus-derived protein can be used.
  • viruses include HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Sever Acute Respiratory Syndro Syndrome).
  • the evaluation method of the present invention As described later, for example, it is possible to identify an antigen epitope peptide, synthesize the peptide, and use the synthesized peptide as a vaccine against a viral infection. Therefore, the evaluation method of the present invention can be used as a vaccine development system for viral infections.
  • the prepared vector is introduced into the antigen-presenting cell of the present invention having antigen-presenting ability.
  • the antigen-presenting cell of the present invention expresses a T cell costimulatory factor and an MHC class I molecule, and all or part of the protein expressed in the cell is placed on the MHC class I molecule. It is a cell derived from an established cell line capable of presenting an antigen.
  • Antigen presenting cells have conventionally used DC or the like. Peripheral blood force DC obtained cannot be proliferated in small numbers, so it is difficult to secure the number to be used as antigen presenting cells. There are many cases. In addition, when isolating peripheral blood isostatic DC progenitor cells, it is necessary to induce differentiation using site force in. In contrast, the antigen-presenting cell of the present invention is advantageous in that, for example, it is easy to proliferate and it is not necessary to induce differentiation, so that preparation of the antigen-presenting cell is easy.
  • the antigen-presenting cell of the present invention is preferably an adherent cell.
  • DC When DC is used as an antigen-presenting cell, there is a problem that the introduction efficiency of the test gene is very low.
  • Antigen When the display cell is an adherent cell, for example, there is an advantage that the vector introduction efficiency by the ribofusion method or the like is improved.
  • the antigen-presenting cells are attached to the bottom surface of a shear or the like, so that the pulse efficiency to lymphocytes is increased.
  • the antigen-presenting cell of the present invention is preferably derived from a tumor cell-derived cell line.
  • tumor-derived cells are not easily established, they can always grow semipermanently and their properties are stable.
  • a cell line obtained by modifying a tumor cell-derived cell line so as to express an MHC class I molecule or a T cell costimulatory factor may be used as the antigen-presenting cell of the present invention.
  • Such antigen-presenting cells of the present invention can be prepared by appropriately using conventionally known methods. For example, reference can be made to the literature [Anal Biochem. 1993 Feb 1; 208 (2): 352-6. Maximal expression of Recombinant cDNAs IN COS cell for use IN expression cloning. Kluxen FW, Lubbert H.].
  • the antigen-presenting cell of the present invention can be prepared using the following procedure.
  • MHC class I molecules and T cell costimulatory factors are expressed. Amplify the gene of the molecule to obtain cDNA, and then amplify it by PCR and incorporate it into an appropriate expression vector.
  • the expression vector is introduced into the cell line to be used, cultured in a medium, and then selected in a medium containing antibiotics.
  • the antigen-presenting cell of the present invention include the MDA-CD80 cell line.
  • the MDA-CD80 cell line refers to a tumor cell line derived from a breast cancer expressing MHC class I molecule, MDA-MB-231, into which a gene encoding CD80 (Cluster Differentiation 80) is introduced.
  • the MDA-MB-231 cell line can be obtained, for example, from ATCC.
  • TUHR10TKB cell line derived from renal cancer JR-st cell line derived from gastric cancer, and the like.
  • T cell costimulatory factor examples include CD80 (B7.1) molecule and CD86 (B7.2) molecule. These molecules are one of the molecules that transmit signals that activate the immune function of lymphocytes. When these molecules bind to the CD28 molecule on the surface of lymphocytes, the lymphocytes are activated and CTLs are activated. Be guided. In the antigen-presenting cell of the present invention, it is more preferable that either CD80 molecule or CD86 molecule is expressed. CD 80 and CD86 molecules can be expressed simultaneously.
  • the gene encoding the test protein is introduced into the antigen-presenting cell by a vector, it is preferable to introduce a gene encoding ubiquitin in addition to the gene encoding the test protein so that it can be expressed. More preferably, a gene encoding a fusion protein of the test protein and ubiquitin is preferably introduced so as to allow expression. By doing so, the transition to the antigen presentation pathway is promoted, the antigen presentation ability is improved, and the CTL induction ability is improved accordingly. As a result, it is possible to evaluate the inducibility of CTL with high sensitivity, that is, to detect the presence of a highly sensitive epitope peptide.
  • ubiquitin is a protein having a strength of 76 amino acid residues, and binds to a target protein to serve as a marker for degradation. Further, ubiquitin is added to the protein to which ubiquitin is bound, and the target protein is added with a polyubiquitin chain. This is a signal for degradation, and the target protein is rapidly degraded by intracellular proteasomes.
  • the method for introducing the vector into the antigen-presenting cell is not particularly limited, and a general method can be used.
  • a general method can be used.
  • the calcium phosphate method, the lipofussion method, the DEAE (dimethylaminoethyl) dextran method, the electo-poration method, the microinjection method and the like are preferable from the viewpoint of high power introduction efficiency.
  • the lipofusion method is a method in which a complex of ribosome, which is a lipid bilayer vesicle, and DNA to be introduced is formed, and the target gene is introduced into the cell by phagocytosis or membrane fusion.
  • lymphocytes are co-cultured with antigen-presenting cells into which a gene encoding a test protein has been introduced.
  • antigen-presenting cells are cultured to allow antigen presentation.
  • the culture period is preferably 24 hours to 48 hours, for example. In the case of longer culture, the ability to induce CTL may decrease.
  • antigen-presenting cells that have been presented with antigens are also called stimulator cells. /
  • lymphocytes it is preferable to use human lymphocytes capable of using various lymphocytes.
  • the lymphocyte is preferably a lymphocyte that shares at least one MHC class I molecule with the antigen-presenting cell of the present invention, and more preferably a lymphocyte that matches the MHC class I molecule.
  • the method for collecting lymphocytes is not particularly limited, and examples thereof include a method for recovering peripheral blood mononuclear cells (Peripheral Blood Mononuclear Ce 11, hereinafter also referred to as PBMC) from blood by density centrifugation gradient method.
  • PBMC peripheral blood mononuclear cells
  • lymphocytes used for co-culture with the stimulator cells are also referred to as responder cells (reactive cells).
  • the stimulator cells and the responder cells are mixed and cultured.
  • the temperature condition is, for example, 34 ° C to 38 ° C, preferably 37 ° C, and the CO condition is, for example, 2 to 10%.
  • the CO condition is, for example, 2 to 10%.
  • it is in the presence of 5% CO.
  • the culture period of the co-culture is preferably about 5 to 7 days. If cultured for more than 5 days, CTL can be induced, and if cultured for a long time without applying a new stimulus, the cell may die.
  • AIM-V medium manufactured by Invitrogen
  • RPMI-1640 Medium manufactured by Invitrogen
  • Dulbecco's modified Eagle medium manufactured by Invitrogen
  • TIL manufactured by Immunobiological Laboratories, Inc.
  • epidermal keratinocyte medium manufactured by Kojin Bio Inc.
  • Iskov medium manufactured by Invitrogen
  • Commercially available media used for cell culture can be used. If necessary, 5-20% urine serum, fetal calf serum (hereinafter also referred to as FCS), human plasma, and the like may be added. Also, if necessary, you can add various site strengths.
  • the confirmation method is not particularly limited, and is a force that can be applied to a conventionally known method.
  • the antigen epitope peptide sequence is divided, it is a tetramer of MHC class 1 / antigen peptide complex.
  • CTL quantification method using tetramer can be applied.
  • ELISPOT method Enzyme-Linked Immunospot Assay
  • a method for measuring intracellular site force in (interferon ⁇ ), etc. can be applied.
  • the antigen protein screening method of the present invention comprises a protein containing an antigenic epitope peptide that specifically induces CTL (hereinafter referred to as an antigen protein) by using the evaluation method of the present invention. It is also a screening method including a step of identifying. With such a method, for example, a protein containing an antigenic epitope peptide can be easily searched from various cancer cell-derived proteins and virus proteins. Further, the identified protein can be targeted for identification for identification of a new antigen epitope peptide, for example, as described later.
  • the method for screening an antigenic epitope peptide of the present invention comprises the step (i) and the step
  • test protein is identified by the screening method for an antigenic protein of the present invention, which may be a known antigenic protein. It may be a novel antigen protein.
  • the test protein is preferably the latter antigen protein.
  • the step (A) is a part of a known antigen protein whose antigen protein or antigen epitope peptide is unknown, for example, identified by the antigen protein screening method of the present invention.
  • This is a step of performing the evaluation method of the present invention using various partial peptides as test proteins (hereinafter also referred to as test partial peptides), and identifying partial peptides containing the antigenic epitope peptides from the test partial peptides.
  • test partial peptides various partial peptides as test proteins
  • test partial peptides partial peptides as test proteins
  • identifying partial peptides containing the antigenic epitope peptides from the test partial peptides By performing such a method by changing the length or cleavage site of the test partial peptide introduced into the antigen-presenting cell, it becomes possible to narrow down the antigen epitope peptide, and finally, the antigen epitope The peptide can be identified.
  • test partial peptides are synthesized based on the antigen protein identified by the antigen protein screening method of the present invention, and the synthesized test partial peptide is converted to the antigen of the present invention.
  • the step of co-culturing with lymphocytes after contacting with the presenting cells or in contact with them, examining the induction efficiency of specific CTLs in the lymphocytes, and identifying the antigen epitope peptide from the test partial peptide is there.
  • the number of amino acid residues of an antigen epitope peptide is generally 9 to 11 residues.
  • the kit of the present invention is an evaluation kit used in the method for evaluating CTL inducibility of the present invention, and comprises the antigen-presenting cell of the present invention.
  • the kit of the present invention is a screening kit for use in the screening method for the antigen protein and Z or antigen epitope peptide of the present invention, comprising the antigen-presenting cell of the present invention. Let's say. Conventionally, in order to evaluate the ability to induce CTL, it is necessary to prepare antigen-presenting cells each time, and such a kit is difficult.
  • kits can be prepared, whereby a simple and highly accurate method for evaluating the ability to induce CTLs and a method for screening antigen proteins and Z or antigen epitope peptides. It becomes possible.
  • the kit of the present invention can be used for culture. Necessary items such as a culture medium and a reagent can be included.
  • the method for producing an antigenic epitope peptide having the ability to induce CTLs of the present invention includes a step of identifying an antigenic epitope peptide by the method for screening an antigenic epitope peptide of the present invention, and other steps. Is not particularly limited. It is preferable that the production method of the present invention further includes a step of separating the antigenic epitope peptide identified as the protein force and a step of synthesizing the identified antigenic epitope peptide.
  • the MDA-CD80 cell line which is a cell line modified from the MDA-MB 231 cell line so as to express human CD80 molecule constitutively, was established by the following method.
  • PCR is performed using LATaq DNA polymerase (Takara) for 3 minutes at 94 ° C, then for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C. The reaction was performed for 30 cycles as a cycle, and finally at 72 ° C for 5 minutes.
  • the DNA fragment specifically amplified by the PCR method was excised by 1.5% agarose gel electrophoresis, It was treated with restriction enzymes Hindlll and Xbal, and inserted between the Hindlll site and Xbal site of plasmid pRcZCMV (Invitrogen) to obtain pRcZCMV-CD80.
  • MDA—MB—231 cells were mixed with a 10% FCS (CELLect).
  • the cells were cultured in RPMI 1640 medium containing Gold Fetal Bovine Serum (manufactured by ICsN Biomedicals, Inc.) for 4 to 6 hours. 6 ⁇ g of the pRcZCMV-CD80 was introduced into the cells by the ribofusion method. 48 hours after introduction, it was confirmed that the CD80 molecule was transiently expressed using a flow cytometer (EPICS XL / MCL, manufactured by Beckman Coulter, Inc.). It was also confirmed. At the same time, the medium was changed to a medium in which the antibiotic G418 was added to the medium, and cells having the plasmid integrated into the chromosome were selected. Furthermore, the selected cells were also clotted by single cell force by the limiting dilution method to obtain a cell line MDA-CD80 that stably expresses human CD80 molecules.
  • the vertical axis represents the number of cells, and the horizontal axis represents the intensity of fluorescence (expression frequency of CD80 molecule) in one cell.
  • the white mountain represents the control (MDA-MB-231) cells, and the black mountain represents the MDA-CD80 cells.
  • FACS confirmed that the CD80 molecule was expressed on the MDA-CD80.
  • lane a is a marker
  • lane b is MDA-MB-231
  • lane c is MDA-CD80.
  • RT-PCR was performed using RNA extracted from cells expressing BMLF1! / And a cDNA library was prepared.
  • the BMLF1 is derived from EBV (Epstein-Barr Virus) It is a protein.
  • the BMLFl gene fragment was selected as much as possible from the prepared cDNA library, and the obtained BMLF1 fragment was amplified by PCR. The amplified gene fragment was found at 1,317 bp.
  • pTracer TM SV40 (trade name, manufactured by Invitrogen, hereinafter also referred to as pTracer) and the ubiquitin gene (hereinafter also referred to as Ub) were cleaved with restriction enzymes Aflll and Kpnl to restrict ⁇ racer. Ub was inserted between the enzyme cleavage sites to create pTracer-Ub.
  • pTracer-Ub and the BMLF1 fragment were cleaved with restriction enzymes Kpnl and Notl, and BMLF1 was inserted between the restriction enzyme cleavage sites of pTracer-Ub to prepare pTracer-Ub-BMLF1.
  • the pTracer and the BMLF1 fragment are cleaved with restriction enzymes Kpnl and Notl, and BMLF1 is inserted between the restriction enzyme cleavage sites of pTracer, p
  • Tracer-BMLF 1 was prepared.
  • MDA—CD80 cell line 1 x 10 6 cells of MDA—CD80 cell line was cultured on 6-well plate (Sumitomo Bakelite) for 4-12 hours, and then pTracer—Ub—BMLFl (4 ⁇ g) was added by lipofusion method. To the MDA-CD80 cells.
  • a blood cell centrifuge (Lymphoprep, manufactured by AXIS—SHIELD PoC AS)
  • PBMCs were prepared by a centrifugation method using) and used as responder cells.
  • the stimulator cells (5 ⁇ 10 5 cells) and the responder cells (2 ⁇ 10 6 cells) were mixed and cultured at 37 ° C. under 5% CO for 7 days. At this time, IL-2 (PROLEUKIN,
  • CHIRON CHIRON
  • FIGS. Fig. 3A is an example of the result of Mock treatment in which only pT racer is introduced into MDA-CD80.
  • Fig. 3B is an example of the result of introduction of p Tracer-BMLF1 into MDA-CD80.
  • Fig. 3C is an example of MDA- This is an example of the result of introducing pTracer-Ub-BMLFl into CD80.
  • 3A to C the horizontal axis shows 1S FITC-CD8 signal, and the vertical axis shows PE-Tetramer signal. As shown in FIG. 3A and FIG.
  • MDA-CD80 cell line of 1 ⁇ 10 6 cells (prepared in Example 1) was cultured on 6-well plates for 4-12 hours.
  • RT-PCR was performed using RNA extracted from cells expressing Marti! /, And cDN
  • Marti is an antigenic protein of melanoma (malignant melanoma).
  • Marti gene fragments selected from the cDNA library prepared above were selected and Marti obtained.
  • the gene fragment was amplified by PCR.
  • the amplified gene fragment was 357 bp.
  • MDA-CD80 cells into which pTracer-Ub-Martl was introduced were treated with 100 ⁇ g of mitomycin per 1 ⁇ 10 6 cells for 30 minutes to form stimulator cells.
  • a blood cell centrifuge (Lymphoprep, manufactured by AXIS—SHIELD PoC AS)
  • PBMCs were prepared by a centrifugation method using) and used as responder cells.
  • the stimulator cells (5 ⁇ 10 5 cells) and the responder cells (2 ⁇ 10 6 cells) were mixed and cultured at 37 ° C. under 5% CO for 7 days. At this time, IL-2 becomes lOOUZml
  • AIM-V medium supplemented with 10% FCS was used.
  • FIGS. Figure 4A shows MDA—CD80 with pT
  • Fig. 4B shows an example of the result of introducing p Tracer-Marti into MDA-CD80
  • Fig. 4C shows the result of introducing pTracer Ub-Marti into MDA-CD80.
  • the horizontal axis represents the FITC-CD8 signal
  • the vertical axis represents the PE-Tetramer signal.
  • FIG. 4A and FIG. 4B it was confirmed that by introducing a gene encoding a full-length protein derived from tumor cells into MDA-CD80, antigens were presented and specific CTLs could be induced.
  • Fig. 4C when a fusion gene of ubiquitin and an antigenic peptide is introduced, the specific CTL induction 'detection efficiency power 2. 7 times improvement was confirmed.
  • BMLF1 By introducing the BMLF1 gene into MDA—CD80 cells to induce CTL and measuring interferon ⁇ (hereinafter also referred to as IFN—y) produced by the induced CTL, BMLF
  • the stimulator cells (5 ⁇ 10 5 cells) and the responder cells (2 ⁇ 10 6 cells) were mixed and cultured at 37 ° C. and 5% CO for 7 days. At this time, IL-2 becomes lOOUZml
  • As the medium AIM-V medium supplemented with 10% FCS was used.
  • the stimulator cells are MDA-CD80 introduced with pTracer-Ub-BMLF 1 and MDA-CD80 introduced with pTracer.
  • responder cells were collected and washed with PBS (-). As described above, since the stimulator cells have been treated with mitomycin in advance, only about responder cells remain in the collected cells after 7 days of culture.
  • the CTL induced by the first stimulator cell stimulation reduces the amount of IFN-y produced during the 7-day culture of IFN- ⁇ .
  • a second stimulation was performed.
  • the first stimulation already induced CTL Because it is being performed, the second stimulation may be brief.
  • stimulator cells for example, ubiquitin antigen MDA-CD80 in which a protein fusion protein is introduced using pTracer or the like can be used
  • the collected responder cells (2 ⁇ 10 5 cells) were suspended in 100 ⁇ l of AIM-V medium supplemented with 10% FCS.
  • MDA-CD80 (1 ⁇ 10 5 cells) pulsed with epitope peptide was suspended in AIM-V medium supplemented with 10% FCS at 100 / z 1 and prepared.
  • the responder cells and the stimulator cells were mixed in a 96-well round bottom plate (manufactured by Sumitomo Beichikrite), and 50 UnitZml IL-2 and 40 ⁇ gZml BefeldinA (manufactured by Sigma Chemicals) were mixed. 4% at 37 ° C and 5% CO.
  • the BefeldinA is a reagent that inhibits the release of cytodynamic force in the cell. Therefore, IFN- ⁇ produced in the cell is accumulated in the cell.
  • IntraPrep Reagent 1 cell membrane permeabilization reagent, manufactured by Beckman Coulter, Inc.
  • the IntraPrep Reagent is a drug for fixing cells. The sample was vortexed to mix well and left at room temperature for 15 minutes. The cells were collected, washed with PBS (-), and suspended in 100 1 PBS (-). Then, 1001 IntraPrep Reagent 2 (cell membrane permeabilization reagent, manufactured by Beckman Coulter, Inc.) was added and allowed to stand at room temperature * location for 5 minutes.
  • the IntraPrep Reagent 2 is a reagent for making a hole in the fixed cell surface so that the antibody can react with intracellular IFN-y.
  • FIG. 5 shows that only pTracer is installed on MDA—CD80.
  • Fig. 5B shows an example of the result of introducing pTracer-Ub-BMLF1 into MDA-CD80.
  • the horizontal axis represents the FITC-CD8 signal, and the vertical axis represents the IFN- ⁇ signal.
  • MDA-CD80 with pTracer—Ub—BMLF 1 is used for the first stimulation, compared to the case where only IFN— ⁇ -quantity pTracer produced is introduced. It was confirmed that the price was getting higher. That is, it was confirmed that by introducing the BMLF1 gene, antigen presentation was performed and specific CTLs were induced.
  • the present invention is useful, for example, in the field of searching for novel antigen epitope peptides, and in the medical field including immune cell therapy for the treatment Z prevention of cancer, infectious diseases and the like.
  • SEQ ID NO: 1 primer CD80 F

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Abstract

L’invention porte sur un procédé permettant d’évaluer rapidement et de façon pratique une capacité d’induction CTL spécifique dans une protéine. Ce procédé d’évaluation de capacité d’induction CRL spécifique à une protéine comprend la phase de transfert d’un gène codant la protéine de test dans des cellules présentant un antigène afin de permettre l’expression de la protéine, la phase de mise en culture des cellules présentant un antigène, dans lesquelles le gène a été transféré, avec des lymphocytes, et la phase d’examen de l’efficacité d’induction CTL dans les lymphocytes, où l’on utilise comme cellules présentant un antigène, des cellules provenant d’une ligne de cellules établies exprimant un costimulateur cellulaire T et une molécule MHC de classe I, capables de présenter comme antigène toute la protéine exprimée dans les cellules ou une partie de celle-ci. Selon ce procédé, on peut cribler rapidement et de façon pratique une protéine antigène ou un peptide épitope antigène ayant une capacité d’induction CTL spécifique, ce qui permet alors de rechercher un nouveau peptide épitope antigène.
PCT/JP2005/017724 2004-10-01 2005-09-27 Procédé pour évaluer la capacité d’induction ctl et procédé de criblage utilisant celui-ci WO2006040920A1 (fr)

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JP2005245430A (ja) * 2003-08-25 2005-09-15 Medeinetto:Kk Ｃｔｌの誘導方法

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Publication number Priority date Publication date Assignee Title
JP2005245430A (ja) * 2003-08-25 2005-09-15 Medeinetto:Kk Ｃｔｌの誘導方法

Non-Patent Citations (4)

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Title
MANGINO G ET AL: "Presentation of native TROP-2 tumor antigens to human cytotoxic T lymphocytes by engineered antigen-presenting cells.", INT J CANCER., vol. 101, no. 4, 2002, pages 353 - 359, XP002994945 *
OKADA N ET AL: "Efficient antigen gene transduction using Arg-Gly-Asp fiber-mutant adenovirus vectors can potentiate antitumor vaccine efficacy and maturation of murine dendritic cells.", CANCER RES., vol. 61, no. 21, 2001, pages 7913 - 7919, XP002994946 *
SCHOENBERGER SP ET AL: "Efficient direct priming of tumor-specific cytotoxic T lymphocyte in vivo by an engineered APC.", CANCER RES., vol. 58, no. 14, 1998, pages 3094 - 3100, XP002971473 *
ZHU E ET AL: "Specific CTL response induced by dendritic cells transfected with total tumor RNA.", BLOOD., vol. 96, no. 11, 2000, pages 28A, XP002994947 *

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