JP2006129832A - CATHEPSIN H PROTEIN-DERIVED CD8+ CYTOTOXIC T-LYMPHOCYTE mHA EPITOPE PEPTIDE AND APPLICATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CD8<SP>+</SP>cytotoxic T-lymphocyte epitope peptide useful for treating hematogenous organ tumor or solid tumor left or relapsed after hematopoietic stem cell transplantation or for preventing such tumor. <P>SOLUTION: The peptide is a partial peptide of Cathepsin H protein, being a minor histocompatibility antigen(mHA) epitope peptide recognizable by CD8<SP>+</SP>cytotoxic T-lymphocyte through binding to a human leukocyte antigen(HLA). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention is a partial peptide of Catthepsin H protein and is recognized by CD8 ⁺ cytotoxic T lymphocyte (hereinafter referred to as CTL) by binding to human major histocompatibility antigen (HLA). Minor histocompatibility antigen (mHA) epitope peptide, hematopoietic tumor or solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation using the peptide, or hematopoietic tumor or solid after allogeneic hematopoietic cell transplantation Vaccine and passive immunotherapeutic agent for preventing tumor recurrence, method for quantifying CD8 ⁺ cytotoxic T lymphocytes that damage hematopoietic tumor cells or solid tumor cells, and allogeneic hematopoietic cell transplantation compatibility using the peptide It relates to a diagnostic method.

  Allogeneic hematopoietic stem cell transplantation (hereinafter referred to as HCT) has been established as a treatment that can bring healing to hematopoietic tumors such as leukemia with a poor prognosis only by chemotherapy. More recently, HCT called mini-transplantation has also been attempted for solid tumors, and a tumor reduction effect is being observed in some solid tumors (eg renal cell carcinoma, malignant melanoma). In fact, some solid tumors have been reported to have abnormally expressed genes specific to hematopoietic cells. For example, the Cathepsin H gene is abnormally expressed in gastric cancer cells and malignant black species, and the HA-1 gene is abnormally expressed in lung cancer and the like (for example, see Non-Patent Documents 1 to 3). However, advanced hematopoietic tumors and solid tumors often recur despite HCT, and the prognosis is not always good.

  In HCT, hematopoietic stem cells are provided to patients from a hematopoietic stem cell provider (hereinafter referred to as a donor) compatible with human major histocompatibility antigen (hereinafter referred to as HLA), but the HLA is consistent between the donor and the patient. However, there are many inconsistencies in minor histocompatibility antigen (hereinafter referred to as mHA) derived from various gene polymorphisms, which are mainly targeted to donor-derived T lymphocytes. An immune response occurs in the patient. If this immune response exerts an anti-tumor effect on the hematopoietic tumor or solid tumor of the patient, it will lead to cure of the primary disease and avoid recurrence. This is called the graft-versus-tumor (hereinafter referred to as GVT) effect.

  In patients with recurrence after HCT, this GVT effect is considered inadequate. For this reason, in order to strengthen the action of donor-derived T lymphocytes and increase the GVT effect, treatment may be performed to reduce the amount of immunosuppressive agent used for rejection prevention. In that case, donor-derived T lymphocytes However, cells in the patient's body are regarded as non-self and strongly excluded, and a fatal complication called graft-versus-host disease (GVHD) often occurs. Therefore, there is a demand for an effective treatment that avoids GVHD and attempts to actively extract and use only the GVT effect.

The main immunocompetent cells that control the recurrence of hematopoietic or solid tumors after HCT are CD8 ⁺ CTLs derived from donor T lymphocytes. Donor-derived CD8 ⁺ CTL has the ability to recognize and destroy non-self when hematopoietic or solid tumor cells remain in the patient. Therefore, by effectively activating the injury function of CD8 ⁺ CTL, the GVT effect is enhanced and the onset of GVHD can be avoided, and a new hematopoietic tumor or solid tumor recurrence prevention method and treatment method at the time of recurrence Is likely to lead to the development of

When CD8 ⁺ CTL recognizes hematopoietic tumor cells and solid tumor cells, it has the following characteristics.
(1) CTL is derived from donor T lymphocytes.
(2) CTL is a partial peptide (hereinafter referred to as mHA epitope) of various proteins (hereinafter referred to as mHA protein) in a patient's hematopoietic tumor cell or solid tumor cell encoded by a gene having a gene polymorphism unique to an individual. Recognizing a peptide consisting of 9 to 10 amino acids encoded by a gene having a genetic polymorphism unique to an individual that differs between a donor and a patient in the Catthepsin H protein, for example, hematopoietic tumor cells Alternatively, solid tumor cells are damaged (destroyed).
(3) This mHA epitope peptide is recognized by binding to HLA on the surface of hematopoietic tumor cells or solid tumor cells and presenting it to CTL.
(4) HLA varies between races and individuals, and if HLA is different, the peptide that becomes the mHA epitope may be different, and thus the presentation to CTL itself may not be possible.

As is clear from what has been described in (1) to (4) above, an mHA protein comprising a peptide that can be an mHA epitope peptide of CD8 ⁺ CTL that damages hematopoietic tumor cells or solid tumor cells is identified, and the epitope Determining the amino acid sequence of a peptide without excess or deficiency diagnoses whether the donor can have an effective GVT effect in patients receiving or receiving HCT, and in performing specific immunotherapy. It can be an essential confirmation item.

However, HLA type (HLA-A24 type, 64%; HLA-A33 type, 25%; HLA-A11 type, 22%; HLA-A2.1 type, 21%; HLA-A31 type that many Japanese have. HLA-B44, 24%; HLA-B52, 20%; HLA-B35, 15%; HLA-B54, 14%; HLA-Cw1, 34%; HLA-Cw * 14 , 24%; HLA-Cw * 0304 type, 22%; HLA-Cw * 0702 type, 22%; HLA-Cw * 0801, 21%; HLA-Cw * 1202, 21%, etc.), especially HLA-A33 type For HLA-A31 type, etc., there are very few reports of mHA epitope peptides recognized by CD8 ⁺ CTL, treat hematopoietic tumors or solid tumors remaining or recurring after HCT, or hematopoietic tumors or solids after HCT There is a need to develop mHA epitope peptides that are useful for preventing tumor recurrence.

Choi et al., Oncogene, Vol. 11, No. 9, 1693-8, 1995 Kenny et al., Oncogene 14: 8, 997-1001, 1997 Written by Fujii et al., Transplantation Magazine, Vol. 73, No. 7, 1137-1141, 2002

The present invention relates to a mHA epitope peptide which is a partial peptide consisting of 9 to 10 amino acid residues of Cathespin H protein and which can be recognized by CD8 ⁺ CTL by binding to HLA, allogeneic hematopoietic cells using the peptide Vaccine and passive immunotherapeutic agent, hematopoietic tumor cell or solid for treating hematopoietic tumor or solid tumor remaining or recurring after transplantation or preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation It is an object of the present invention to provide a method for quantifying CD8 ⁺ CTL that damages tumor cells, and a diagnostic method for compatibility with allogeneic hematopoietic cell transplantation using the peptide.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found an mHA epitope peptide that can be recognized by CD8 ⁺ CTL that damages hematopoietic tumor cells or solid tumor cells, and completed the present invention. It was.

That is, the present invention provides the following (1) to (19).
(1) A minor histocompatibility antigen (mHA) epitope peptide that is a partial peptide of Cathepsin H protein and can be recognized by CD8 ⁺ cytotoxic T lymphocytes by binding to human major histocompatibility antigen (HLA) .
(2) The minor histocompatibility antigen (mHA) epitope peptide according to (1), wherein the partial peptide of Cathepsin H protein has 5 to 20 amino acid residues.

(3) The minor histocompatibility antigen (mHA) epitope peptide according to (2), wherein the partial peptide of Cathepsin H protein has 9 to 10 amino acid residues.
(4) The partial peptide of the Cathepsin H protein is a partial peptide comprising the 19th, 39th or 82nd amino acid in the amino acid sequence represented by SEQ ID NO: 1 (1) to (3 ) Any one of the peptides.

(5) The peptide according to any one of (1) to (4), wherein the HLA is HLA-A33.
(6) The peptide according to (5), which is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4.
(7) The peptide according to any one of (1) to (4), wherein the HLA is HLA-A31.

(8) The peptide according to (7), which is a peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 4 to 7.
(9) The peptide according to any one of (1) to (4), wherein HLA is HLA-A2.
(10) The peptide according to (9), which is a peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 8 to 15.

(11) The peptide according to any one of (1) to (10), wherein the CD8 ⁺ cytotoxic T lymphocyte damages hematopoietic tumor cells or solid tumor cells.
(12) The peptide according to (11), wherein the hematopoietic tumor is leukemia, myelodysplastic syndrome, malignant lymphoma, or multiple myeloma.
(13) The peptide according to (11), wherein the solid tumor is renal cell carcinoma, malignant melanoma, breast cancer, stomach cancer, lung cancer or prostate cancer.

(14) Treating a hematopoietic tumor or solid tumor that has remained or recurred after allogeneic hematopoietic cell transplantation, comprising as an active ingredient the peptide according to any one of (1) to (13) or an antigen-presenting cell pulsed with the peptide. Or a vaccine for preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation.
(15) Allogeneic hematopoiesis comprising CD8 ⁺ cytotoxic T lymphocytes obtained by stimulating peripheral blood lymphocytes with the peptide according to any one of (1) to (13) or antigen-presenting cells pulsed with the peptide A passive immunotherapeutic agent for treating hematopoietic tumor or solid tumor remaining or recurring after cell transplantation, or preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation.

(16) A major histocompatibility complex prepared from the peptide according to any one of (1) to (13) and / or a major histocompatibility complex-tetramer and a peripheral blood lymphocyte are reacted, and the major tissue Compliant antigen complexes and / or major histocompatibility complex - tetramer CD8 ⁺ cytotoxic T lymphocytes to form a conjugate bound obtained by isolating from the conjugate CD8 ⁺ cytotoxic T lymphocytes A passive immunotherapeutic agent for treating hematopoietic tumor or solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation, or preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation, comprising:

(17) A major histocompatibility complex-labeled magnetic bead prepared by reacting a major histocompatibility complex-labeled magnetic bead prepared from the peptide according to any one of (1) to (13) with peripheral blood lymphocytes. A hematopoietic tumor that has remained or recurred after allogeneic hematopoietic cell transplantation, comprising CD8 ⁺ cytotoxic T lymphocytes obtained by forming a conjugate with CD8 ⁺ cytotoxic T lymphocytes bound thereto Alternatively, a passive immunotherapeutic agent for treating solid tumors or preventing recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation.

(18) (1) to stimulate the peripheral blood with the peptide of any one of (13) to obtain a CD8 ⁺ cytotoxic T lymphocytes, cytokine the CD8 ⁺ cytotoxic T lymphocytes produce and / Alternatively, a method for quantifying CD8 ⁺ cytotoxic T lymphocytes that damage hematopoietic tumor cells or solid tumor cells, characterized by measuring chemokines.

(19) A major histocompatibility complex and / or major histocompatibility complex-tetramer is prepared from the peptide according to any one of (1) to (13), and the major histocompatibility complex and / or major A method for quantifying CD8 ⁺ cytotoxic T lymphocytes which damage hematopoietic tumor cells or solid tumor cells in the peripheral blood, wherein the histocompatibility complex-tetramer is reacted with peripheral blood.
(20) Adaptability to allogeneic hematopoietic cell transplantation, including genotyping of donor- and patient-derived DNA in allogeneic hematopoietic cell transplantation using the base sequence of the region containing the polymorphic site of the Catthepsin H gene shown in SEQ ID NO: 2 Diagnostic method.

(21) A donor and patient-derived DNA in allogeneic hematopoietic cell transplantation is amplified by the PCR method using the base sequence of the region containing the polymorphic site of the Catthepsin H gene shown in SEQ ID NO: 2, and the obtained PCR amplification product is donor And the allogeneic hematopoietic cell transplantation compatibility diagnostic method according to (20), comprising genotyping between patients.
(22) The allogeneic hematopoietic cell transplantation compatibility diagnostic method according to (20) or (21), wherein the base sequence of the region containing the polymorphic site of the Cathepsin H gene has a base length of 18 to 30 mer.

(23) The base sequence of the region containing the polymorphic site of the Catthepsin H gene shown in SEQ ID NO: 1 is the base sequence encoding the mHA epitope peptide according to any one of (20) to (23) The diagnostic method according to.
(24) An isolated antigen-presenting cell pulsed with at least one peptide of any one of (1) to (13) and bound to the HLA expressed on the surface.
(25) A method for activating antigen-presenting cells, comprising culturing antigen-presenting cells in vitro together with at least one peptide of the peptide according to any one of (1) to (13), An activation method wherein the antigen-presenting cells can stimulate CD8 ⁺ cytotoxic T lymphocytes.

(26) A method for preparing CD8 ⁺ cytotoxic T lymphocytes that can be used for the prevention and treatment of hematopoietic tumors or solid tumors by any of the following methods (a) to (c).
(a) A method comprising stimulating peripheral blood lymphocytes in vitro using the peptide according to any one of (1) to (13) or an antigen-presenting cell pulsed with the peptide,
(b) A major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the peptide according to any one of (1) to (13) is reacted with peripheral blood lymphocytes, and the major tissue Compliant antigen complexes and / or major histocompatibility complex - the CD8 ⁺ cytotoxic T lymphocytes to form a conjugate bound to isolate CD8 ⁺ cytotoxic T lymphocytes from the conjugate to tetramer And a method comprising
(c) The main histocompatibility complex-labeled magnetic beads prepared from the peptide according to any one of (1) to (13) are reacted with peripheral blood lymphocytes, and the main histocompatibility complex-labeled magnetic beads are reacted. A method in which CD8 ⁺ cytotoxic T lymphocytes are bound to each other and a CD8 ⁺ cytotoxic T lymphocyte is isolated from the conjugate (27) (26). Isolated CD8 ⁺ cytotoxic T lymphocytes that can be used for the prevention and treatment of hematopoietic or solid tumors.

According to the present invention, it is possible to provide a mHA epitope peptide that is a partial peptide of Cathesin H protein and that can be recognized by CD8 ⁺ CTL by binding to human major histocompatibility antigen (HLA). The peptide can also be used to treat or prevent hematopoietic or solid tumors in patients who have undergone HCT. Furthermore, CD8 ⁺ CTL that damages hematopoietic tumor cells or solid tumor cells in the patient's body after HCT can be quantified. It is also possible to provide a diagnostic method for compatibility with allogeneic hematopoietic cell transplantation using the peptide.

The present invention will be described in further detail.
1. MHA epitope peptide that can be recognized by CD8 ⁺ CTL Peptide in the present invention is a peptide having linear activity and is a linear amino acid that is bound to each other by peptide bond between α-amino group and carboxyl group of adjacent amino acid residues. Means a molecular chain. Peptides are not meant to be of a specific length and can be of various lengths. Further, it may be in an uncharged or salt form, and may be modified by glycosylation, amidation, phosphorylation, carboxylation, phosphorylation or the like in some cases. Furthermore, one or several (for example, 1 to 8, preferably 1) unless the physiological activity and immune activity of the epitope of the present invention are substantially altered and do not have harmful activity when administered. Peptides in which insertion, addition, substitution, etc. of (˜3) amino acids have occurred are also included in the present invention. For example, those having an additional amino acid sequence at the N-terminus or C-terminus of the peptide are also included. In addition, the peptide of the present invention can be used in the form of a complex to which saccharides, polyethylene glycol, lipids and the like are added, a derivative with a radioisotope, or a polymer.

In addition, the mHA epitope peptide that can be recognized by CD8 ⁺ CTL in the present invention is presented by binding to HLA on the surface of hematopoietic tumor cells or solid tumor cells, and is a T cell antigen receptor of donor-derived CD8 ⁺ CTL A partial peptide of mHA protein present in hematopoietic tumor cells or solid tumor cells that can be recognized by CD8 ⁺ CTL by binding to (TCR) and encoded by a gene having an individual-specific genetic polymorphism Means a peptide containing a polymorphic site. The peptide preferably consists of 5 to 20 amino acids, more preferably 5 to 15, 7 to 12 or 8 to 11 amino acids, particularly preferably 9 to 10 amino acids. Thus, the peptide activates the cellular immune mechanism of CD8 ⁺ CTL that can directly damage (destroy) and eliminate certain types of hematopoietic or solid tumor cells of HLA that express mHA protein It is an antigen group. CD8 ⁺ CTL means CD8 ⁺ cytotoxic T lymphocytes that express CD8, which is one of the surface antigen molecules present on human lymphocytes. FIG. 1 shows the mechanism.

  In the present invention, hematopoietic tumors include tumors arising in hematopoietic tissues or cells such as leukemia, myelodysplastic syndrome, malignant lymphoma, multiple myeloma, and the like. In the present invention, the solid tumor includes any solid tumor as long as it abnormally expresses the Cathepsin H gene. Specifically, renal cell carcinoma, malignant melanoma, breast cancer, stomach cancer, lung cancer, prostate Examples include cancer.

  Possible candidates for the mHA protein are various proteins that exist only in hematopoietic tumor cells or solid tumor cells and are encoded by genes having genetic polymorphism unique to the individual. Among these, since the gene is expressed only in hematopoietic cells and some solid organs, the Catepsin H protein is particularly useful for identifying the mHA epitope peptide of the present invention.

  Cathepsin H protein is a cysteine proteolytic enzyme of lysozyme and is important for the degradation of proteins in lysozyme. It has been reported that the expression of a gene encoding this protein is enhanced in prostate cancer (Waghray et al., J Biol Chem, Vol. 277, 11533-11538, 2002). The expression of this gene is observed not only in hematopoietic cells but also in the lung, kidney, and liver. However, CTL specific for the mHA epitope peptide of the present invention does not damage at least skin fibroblasts and bone marrow stromal cells. Expression of mHA and the generation of mHA epitopes are not likely to be parallel.

  The base sequence (SEQ ID NO: 1) of the gene encoding Cathespin H protein and the amino acid sequence (SEQ ID NO: 2) of the protein are http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val = 23110954 & db = Nucleotide & dopt = Reported to GenBank.

  The 11th polymorphism (SEQ ID NO: 3) of the amino acid sequence of Catthepsin H protein can be found at http: //www.ncbi.National Center for Biotechnology Information (NCBI) of National Institute of Health (NIH) in the United States. It can be confirmed by nlm.nih.gov/SNP/snp_ref.cgi?rs=2289702. The eleventh polymorphism of the amino acid sequence is based on the single nucleotide polymorphism of the 126th base of the base sequence of the Cathepsin-H gene (SEQ ID NO: 1) (the 31st base of DNA encoding the Cathepsin-H protein). The wild type is g, whereas the mutant type is A, and the wild type amino acid Gly is mutated to Arg.

  It should be noted that genetic polymorphism unique to an individual means whether or not there is a difference in the sequence between individuals by performing nucleotide sequencing for the Catepsin H gene for DNA obtained from a large number of individuals (for example, 50 people). Search and determine if there is a sequence difference between individuals, determine if it is universal found in multiple individuals, and guess by what percentage of the population each polymorphism exists It is an individual-specific genetic polymorphism that can be done.

  Since the suitable mHA epitope peptides of the present invention differ depending on the HLA type, they can be identified by screening peptides that can bind in each HLA type. Specifically, in the amino acid sequence of Cathepsin H protein, the partial peptide comprising the 11th amino acid has a binding motif of each type of HLA (for example, HLA-A33, A31, A2, etc.), 9 to A collation medium capable of searching for epitope peptides consisting of 10 amino acids, for example, HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/) of BioInformatics & Molecular Analysis Section (BIMAS) published on the Internet. molbio / hla_bind / index.html), and peptides that can be mHA epitope peptides (hereinafter referred to as mHA epitope candidate peptides) can be screened.

  The mHA epitope candidate peptide can be obtained by ordinary chemical synthesis. For example, organic chemical synthesis methods such as solid phase peptide synthesis methods, or DNAs encoding peptides can be prepared and prepared using recombinant DNA technology. Moreover, the synthesis | combination by a commercially available chemical synthesizer (For example, the peptide synthesizer of Applied Biosystems) is also possible.

The obtained mHA epitope candidate peptide can be determined as to whether or not it is an mHA epitope peptide that can be recognized by CD8 ⁺ CTL, for example, by performing any of the methods as shown below.

(1) mHA epitope peptide determination method 1
Suspend lymphocytes isolated from an adult who had previously searched for a Cathepsin H gene polymorphism at a cell concentration of 2 x 10 ⁶ / ml in RPMI1640 medium containing 10% human serum, and isolate the lymphocytes from the same person in advance. Mix with cultured dendritic cells (1 × 10 ⁵ / ml) or activated B cells (1 × 10 ⁶ / ml) and add 1 μg / ml of any one of mHA epitope candidate peptides. Add at a concentration of. Incubate for 10 days at 37 ° C in a carbon dioxide oven. On the 10th day, IL-2 is added, and then the stimulation with the candidate peptide and IL-2 is repeated once a week to induce CD8 ⁺ CTL specific for the polymorphic site of Catepsin H protein. Whether or not the mHA epitope candidate peptide stimulates the CD8 ⁺ CTL thus induced is determined by an Elispot assay or the like. Elispot assay is reported in Miyahira Y, et al., Journal of Immunological Methods, 181: 45-54, 1995, etc. As a result of the determination, a candidate peptide in which stimulation to the induced CD8 ⁺ CTL is recognized is determined as the mHA epitope peptide of the present invention.

(2) mHA epitope peptide determination method 2
A peptide library of a partial peptide containing the 11th amino acid in the amino acid sequence of Cathepsin H protein is synthesized by a conventional method. The number of amino acid residues of the partial peptide is preferably 5 to 20, more preferably 5 to 15, 7 to 12, or 8 to 11, and particularly preferably 9 to 10. From the synthesized peptide, an mHA epitope peptide that reacts with CD8 ⁺ CTL is selected by the Elispot method or intracellular IFNγ-producing cell quantification method to obtain the epitope peptide of the present invention.

  The peptides having the amino acid sequences of SEQ ID NOs: 4 to 15 of the present invention have been confirmed as mHA epitope peptides as a result of the above screening and determination methods. The mHA epitope peptides shown in SEQ ID NOs: 4 to 15 of the present invention can be prepared by various conventional peptide synthesis methods.

2. Vaccines The mHA epitope peptides of the present invention can be used as vaccines in active immune peptide vaccine therapy. That is, by administering a vaccine comprising the mHA epitope peptide of the present invention to a patient after HCT, CD8 ⁺ CTL recognizing the peptide is proliferated in the body, which is useful for prevention and treatment of hematopoietic tumors or solid tumors. be able to. As the mHA epitope peptide to be used, one or more peptides corresponding to the patient's HLA and mHA types may be used, or they may be used in combination. For example, when there are a plurality of HLAs capable of presenting an mHA epitope peptide, two or more peptides can be used in combination. In addition, for example, when two or more different mHA epitope peptides (partial peptides having different start sites containing the 11th amino acid) exist as peptides that bind to a specific type of HLA, these two or more peptides Can be used in combination. In addition, vaccines containing antigen-presenting cells (for example, dendritic cells, B cells, macrophages, etc.) pulsed with the mHA epitope peptide of the present invention (hereinafter referred to as mHA epitope peptide pulse cells) can also be used. . Here, the antigen-presenting cell means a cell that expresses HLA to which the peptide can bind on its surface and has CD8 ⁺ CTL stimulating ability, and pulsing means, for example, in a suitable culture solution. This means that the antigen-presenting cells and the peptide are mixed for about 30 minutes to 1 hour. Antigen-presenting cells can be obtained from peripheral blood or umbilical cord blood by a known method. For example, dendritic cells, B cells, macrophages, etc., which are antigen-presenting cells, have antigens specific to each cell on the cell surface, so antigen-presenting cells can be isolated using antibodies against the antigen Alternatively, antigen-presenting cells can be isolated by removing cells other than antigen-presenting cells. At this time, a flow cytometer can also be used. Thus, the antigen-presenting cells pulsed with the mHA epitope peptide have the mHA epitope peptide bound to HLA expressed on the surface thereof, and can stimulate CD8 ⁺ cytotoxic T lymphocytes.

  A vaccine comprising the mHA epitope peptide of the present invention or mHA epitope peptide pulsed cells can be prepared using methods known in the art. For example, as such a vaccine, there are an injection or a solid preparation containing the mHA epitope peptide or mHA epitope peptide pulsed cell of the present invention as an active ingredient. The mHA epitope peptide can be formulated in a neutral or salt form. For example, pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid and tartaric acid. . The mHA epitope peptide or mHA epitope peptide pulse cell of the present invention is pharmaceutically acceptable and is compatible with the peptide or the activity of the cell, for example, water, saline, dextrose, ethanol, glycerol. , DMSO, other adjuvants, etc., or a combination thereof can be used. Furthermore, you may add adjuvants, such as albumin, a wetting agent, and an emulsifier, as needed.

  The vaccine of the present invention can be administered by parenteral administration and oral administration, but parenteral administration is generally preferred. Parenteral administration includes injections such as subcutaneous injections, intramuscular injections, intravenous injections, and suppositories. For oral administration, it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, and cellulose.

The vaccine of the present invention is administered in a therapeutically effective amount. The dose to be administered depends on the subject to be treated and the immune system, and the required dose is determined by the judgment of the clinician. In general, an appropriate dose is 1 to 100 mg for mHA epitope peptide and 10 ⁶ to 10 ⁹ for mHA epitope peptide pulsed cells per patient. In addition, the administration interval can be set according to the subject and purpose.

3. Passive Immunotherapeutic Agent The mHA epitope peptide of the present invention can be used for the preparation of a prophylactic or therapeutic passive immunotherapeutic agent for hematopoietic tumor recurrence after HCT. 1) CD8 ⁺ CTL obtained by stimulating peripheral blood lymphocytes with the peptide 2) Major histocompatibility complex (hereinafter referred to as MHC) prepared from peripheral blood lymphocytes from the peptide or MHC- tetramer reacted to form a MHC or MHC- conjugate CD8 ⁺ CTL is bonded to tetramer, CD8 ⁺ CTL obtained by isolating from the conjugate, or, 3) the peptide of peripheral blood lymphocytes MHC- tetramer was prepared from - reacted with labeled magnetic beads, MHC- tetramer - labeled magnetic beads to CD8 ⁺ CTL is to form a conjugate bound, including CD8 ⁺ CTL, derived isolated from the conjugate, Passive immunotherapy agents can be obtained. Here, the magnetic beads are spherical particles having a diameter of several tens of nanometers to several tens of micrometers having a magnetic material such as iron oxide as a core and the surface being coated with another material. In the present invention, known magnetic beads can be used. The MHC-tetramer can be bound to the magnetic beads by a known method such as adsorption or covalent bonding using a functional group. MHC and MHC-tetramers using mHA epitope peptides can be prepared, for example, as follows. MHC, which is a complex of HLA heavy chain purified from E. coli for protein expression, β2 microglobulin, and the mHA epitope peptide of the present invention, is formed in the buffer. A biotin binding site is added in advance to the C-terminus of the recombinant HLA heavy chain protein, and biotin is added to this site after MHC formation. MHC-tetramers are prepared by mixing commercially available labeled dye streptavidin and biotinylated MHC at a molar ratio of 1: 4. 2 and 3 show the obtained MHC-tetramer and its reaction mechanism, respectively. In each step, it is preferable to perform protein purification by gel filtration.

CD8 ⁺ CTL capable of recognizing the mHA epitope peptide of the present invention contained in the passive immunotherapy agent can be obtained by the following preparation method.
(1) CD8 ⁺ CTL preparation method 1
Lymphocytes are separated from the peripheral blood of the patient after HCT or HCT donor and reacted with MHC or MHC-tetramer at an appropriate concentration prepared as described above at 37 ° C. for 15 minutes. Since CD8 ⁺ CTL bound to the mHA epitope peptide in MHC or MHC-tetramer is stained with a labeled dye, only the stained CD8 ⁺ CTL is isolated using a flow cytometer or the like. Alternatively, it can be reacted with MHC and / or MHC-tetramer solid-phased on a sterile plate or the like in advance. To isolate CD8 ⁺ CTL bound to the mHA epitope peptide in MHC and / or MHC-tetramer immobilized on the plate, wash away other cells that are not bound and then plate Suspend only the remaining CD8 ⁺ CTL in a new culture medium. Since CD8 ⁺ CTL isolated in this way can damage hematopoietic tumor cells or solid tumor cells, it can be stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, or IL-2 for passive immunotherapy. Ensure the required number of cells (see Figure 6).

(2) CD8 ⁺ CTL preparation method 2
The biotinylated MHC prepared as described above is bound to streptavidin-labeled magnetic beads to produce a conjugate (hereinafter referred to as MHC-magnetic beads). MHC-magnetic beads are shown in FIG. Lymphocytes are separated from the peripheral blood of the patient after HCT or HCT donor, and the MHC-magnetic beads having an appropriate concentration are reacted at a lymphocyte: bead ratio of 1:10. When a test tube containing CD8 ⁺ CTL bound to CD8 ⁺ CTL bound to MHC / mHA epitope peptide in MHC-magnetic beads is placed in a magnetic field, CD8 ⁺ CTL bound to the bead is the inner wall of the test tube on the magnet side Sent to. The mechanism is shown in FIG. Thereafter, after washing away other cells, the test tube is removed from the magnetic field, and only the antigen-specific CD8 ⁺ CTL remaining on the inner wall of the test tube is suspended in a new culture medium. Since CD8 ⁺ CTL isolated in this way can damage hematopoietic tumor cells or solid tumor cells, it is stimulated and proliferated with T-cell stimulating drugs such as anti-CD3 antibody, PHA, IL-2, etc. and is necessary for passive immunotherapy Ensure a sufficient number of cells (see FIG. 6).

(3) CD8 ⁺ CTL preparation method 3
Lymphocytes isolated from the peripheral blood of patients after HCT or HCT donors are directly stimulated with the mHA epitope peptide of the present invention or antigen-presenting cells pulsed with the peptide (for example, dendritic cells, B cells, macrophages, etc.) Stimulate with. CD8 ⁺ CTL induced by stimulation is cultured at 37 ° C. for 7 to 10 days in a carbon dioxide thermostat. IL-2 is then added to ensure the number of CD8 ⁺ CTL required for passive immunotherapy by repeating stimulation with mHA epitope peptide and IL-2 or antigen-presenting cells and IL-2 once a week. (See FIG. 6).

Since CD8 ⁺ CTL obtained as described above can damage hematopoietic tumor cells or solid tumor cells, it is suspended in human albumin-containing PBS or the like to prevent relapse of hematopoietic tumor or solid tumor after HCT. Or therapeutic passive immunotherapeutic agent.

4). CD8 ⁺ CD8 ⁺ CTL capable of recognizing mHA epitope peptide quantification invention CTL is, hematopoietic tumors or malignant hematologic malignancies or solid tumors is a patient (primary disease recurrence high risk of solid tumors after HCT is To know whether it is present in the peripheral blood after HCT of patients who have been administered high-potency immunosuppressive agents after HCT) to determine the appropriate use of immunosuppressive agents and to predict the risk of recurrence This is important information for patient management after HCT. The quantification of CD8 ⁺ CTL that recognizes the mHA epitope peptide of the present invention and can damage hematopoietic tumor cells or solid tumor cells can be performed by the following two methods using the mHA epitope peptide of the present invention.

In the first quantification method, interferon gamma (IFNγ) produced by CD8 ⁺ CTL induced by stimulating lymphocytes isolated from peripheral blood after transplantation of HCT patients with the mHA epitope peptide of the present invention, interleukin And the like, and / or a chemokine quantification method. Cytokines and chemokines that can be quantified in the present invention are not limited, and as described above, cytokines include IFNγ, interleukins, colony stimulating factors, etc., and chemokines include CXC chemokines, CC chemokines, C chemokines, CX3C chemokines, Including. A specific method will be described below using IFNγ as an example.

(1) Method 1 (quantitative determination of intracellular IFNγ-producing cells) by cytokine quantification;
Lymphocytes isolated from the peripheral blood of patients after HCT or HCT donors are suspended in RPMI1640 medium containing 10% human serum at a cell concentration of 2 × 10 ⁶ / ml, and the mHA epitope peptide of the present invention at a concentration of 1 μg / ml Add. Furthermore, Brefeldin A etc. which are intracellular protein transport inhibitors are added, and it culture | cultivates at 37 degreeC for 5 to 6 hours in a carbon dioxide thermostat. After culture, the cells are fixed, subjected to membrane permeabilization, and reacted with dye-labeled anti-IFNγ antibody and anti-CD8 antibody. Using a flow cytometer or the like, the percentage of IFNγ positive cells in CD8 ⁺ lymphocytes is quantified.

(2) Method 2 (erypot spot assay) by cytokine quantification;
After coating 96-well MultiScreen-HA plate (Mi11ipore) with anti-IFNγ monoclonal antibody at 4 ° C overnight and washing each well with PBS, lymphocytes separated from the peripheral blood of patients after HCT or HCT donor Slowly. The mHA epitope peptide is put into each well and cultured for 20 hours in a 5% CO ₂ incubator at 37 ° C. On the next day, the plate is washed with PBS supplemented with 0.05% Tween-20 and then reacted in the order of anti-IFNγ rabbit serum and peroxidase-labeled anti-rabbit IgG goat serum for 90 minutes each at room temperature. Furthermore, 0.1M sodium acetate buffer (pH 5.0) containing 3-amino-9-ethylcarbasole (Sigma) and 0.015% H ₂ O ₂ is put into each well and reacted at room temperature for 40 minutes. IFNγ spots are visualized and counted with a stereomicroscope.

(3) Method 3 by cytokine quantification (method of quantifying IFNγ secreted into the culture supernatant);
Peripheral blood of patients after HCT or HCT donors separated from peripheral blood (suspended in RPMI1640 medium containing 10% human serum at a cell concentration of 2 × 10 ⁶ / ml and the mHA epitope peptide of the present invention at a concentration of 1 μg / ml Incubate for 24-48 hours in a carbon dioxide thermostat at 37 ° C. After incubation, collect the supernatant, and measure the IFNγ concentration contained in it in a commercially available ELISA kit (eg, HUMAN IFN gamma ELISA from ENDOGEN) Quantify using.

As a second quantitative method, CD8 ⁺ CTL capable of recognizing mHA epitope peptide and damaging hematopoietic tumor cells or solid tumor cells using MHC-tetramer prepared using mHA epitope peptide of the present invention is used. It can be quantified. The preparation of MHC-tetramer is as described above. The quantification can be performed, for example, as follows. Lymphocytes are isolated from the peripheral blood of patients after HCT or HCT donor, and reacted with MHC-tetramer at an appropriate concentration at 37 ° C for 15 minutes. Since CD8 ⁺ CTL bound to the tetramer is stained with a labeled dye, it is counted using a flow cytometer or the like.

For example, CD8 ⁺ CTLs that damage hematopoietic tumor cells or solid tumor cells in the peripheral blood of patients after HCT can be quantified using MHC-tetramers prepared from mHA epitope peptides as follows.

1. Preparation of MHC-tetramer
(1) Preparation of MHC A large amount of HLA A * 3303 heavy chain and β2 microglobulin are prepared and purified using a recombinant protein expression system using E. coli. An amino acid sequence recognized by biotin ligase is added in advance to the C-terminus of HLA A * 3303 heavy chain. Purified HLA A * 3303 heavy chain and β2 microglobulin were each dissolved in 8M urea. In 200 ml of refolding buffer (pH 8.0; 100 mM Tris-HCl, 400 mM L-arginine-HCl, 2 mM EDTA, 0.5 mM oxidative glutathione, 5 mM reduced glutathione), 12 mg of peptide of SEQ ID NO: 4, HLA A * 3303 heavy chain 18.6 mg and β2 microglobulin 13.2 mg were each injected using a syringe with a 27 gauge needle. After stirring for 48 to 72 hours in a 10 ° C constant temperature bath and promoting the formation of MHC, the refolding buffer containing MHC was dialyzed against 1.8 L of distilled water for 24 hours in a 4 ° C constant temperature bath, and the refolding buffer after dialysis Concentrate to 2 ml using Centriprep 10 (MILLIPORE, Bedford, MA). MHC that flows out around 45 KD molecular weight is isolated by gel filtration chromatography using a Superdex 200 HR (Amersham Pharmacia Biotech AB, Uppsala, Sweden) column.

(2) Preparation of biotinylated MHC Using biotin ligase (AVIDITY, Denver, CO), biotin is bound to a specific site at the C-terminus of HLA A * 3303 heavy chain. MHC to which biotin is added is purified by gel filtration chromatography using a Superdex 200 HR column.

(3) Preparation of MHC-tetramer
PE labeled streptavidin (Molecular Probe, Eugene, OR) and purified biotinylated MHC are mixed at a molar ratio of 1: 4. The MHC-tetramer containing the peptide of SEQ ID NO: 5 flowing out with a molecular weight of about 480 KD is isolated by gel filtration chromatography using a Superdex 200 HR column. Concentrate to about 3 mg / ml using Centricon 10 (MILLIPORE) and store at 4 ° C. As preservatives, Na-Azide, EDTA, Leupeptin, and Pepstatin may be added.

2. Quantification of CD8 ⁺ CTL that damage hematopoietic or solid tumor cells using MHC-tetramer
Peripheral blood lymphocytes (1 × 10 ⁶ ) isolated from patients with HLA-A33 who underwent HCT with a donor incompatible with the mHA epitope peptide presented in HLA-A33 of Cathepsin H protein, 31 days after HCT, Alternatively, lymphocytes (1 × 10 ⁶ ) proliferated after stimulation of peripheral blood lymphocytes of the patient with the peptide shown in SEQ ID NO: 4 for 2 weeks are suspended in 50 μL of PBS containing 2% FCS in a 1.5 ml Eppendorf tube. . To this, 1 μL each of MHC-tetramer containing the peptide of SEQ ID NO: 4 and FITC-labeled anti-CD8 antibody (CALTAG, Burlingame, Calif.) Are added and allowed to stand for 15 minutes in a 37 ° C. thermostat. The cells after the reaction were washed 3 times with 1 ml of PBS containing 2% FCS and then suspended in PBS (1 ml) containing 0.5% paraformaldehyde. CD8 ⁺ CTL bound to the MHC-tetramer is stained with a labeling dye, and tetramer positive cells in FITC-CD8 ⁺ T cells are counted using FACS Calibur (Becton Dickinson), and the ratio is calculated.

As a result, CD8 ⁺ CTL that binds to MHC-tetramer and damages hematopoietic tumor cells or solid tumor cells is stained and migrates to the right. From this, it can be shown that the MHC-tetramer specifically stains CD8 ⁺ CTL which has been stimulated by the mHA epitope peptide of SEQ ID NO: 4 and proliferated.

5. Compatible genetic diagnosis
By using the region containing the polymorphic site of the Cathepsin H gene and carrying out a known PCR method, etc., genetic compatibility diagnosis of donor and patient in HCT therapy is made, and immunotherapy to strengthen GVT Diagnosis of useful combinations can be made. That is, the compatibility between the donor and patient can be diagnosed by typing the single nucleotide polymorphism (SNP) of the Catthepsin-H gene. The nucleotide sequence of at least one oligonucleotide primer and / or probe used in the PCR method includes the polymorphic site of the Catthepsin H gene, and the polymorphic site possessed by the donor and the patient can be confirmed by the PCR method. As long as the number of bases of the oligonucleotide primer and / or probe can be appropriately set within the range of 18 to 30 mer generally used. An example of a base sequence that can be used as the base sequence of such an oligonucleotide primer and / or probe includes a base sequence encoding the mHA epitope peptide of the present invention. Diagnosis includes the following methods.

(1) TaqMan PCR method
PCR primers are designed to amplify the region flanking each polymorphic site of the Cathepsin H gene. Also prepared is a PCR primer prepared by binding a different fluorescent substance (fluorescent reporter dye such as FAM or VIC) and a quencher to a probe specific to the base sequence of the region containing the polymorphic site of the Cathepsin H gene. In addition, when PCR is performed on sample DNA (DNA isolated from approximately 1 ml of patient and donor peripheral blood using a commercially available DNA extraction kit (for example, QIAamp DNA Blood Mini Kit), etc.) Only when a base sequence to be present is present, the corresponding probe is decomposed to generate fluorescence. In the PCR reaction, both sequences are hybridized using stringency conditions that do not hybridize when there is one mismatch between the base sequence of the sample DNA and the base sequence of the probe. Stringency conditions vary depending on the hybridization conditions to be performed, but those skilled in the art should appropriately set conditions such as solvent composition such as temperature, salt concentration, surfactant concentration, etc. based on the hybridization protocol. Can do. By measuring the fluorescence generated, the pattern of the 11th amino acid of the Catthepsin H protein of the donor and patient is read. Whether the mHA epitope peptide is compatible or incompatible by performing typing between donor and patient, and if it is incompatible, is it a useful combination for immunotherapy that enhances GVT? It becomes possible to diagnose whether or not.

(2) Allele-specific PCR method
The primer is designed so that one 3 ′ side of the PCR primer is specific to each base sequence in the region containing the polymorphic site of the Cathespin H gene. The other primer is common to both alleles. When the PCR reaction is performed individually on the sample DNA prepared as described above, amplification occurs in the PCR reaction only when there is a primer that matches the base sequence of the region containing the polymorphic site of the Catthepsin H gene. This makes it possible to determine the compatibility between donors and patients in HCT therapy.

(3) PCR-RFLP method
PCR primers are designed to amplify the region containing the polymorphic site of the Cathepsin H gene. After the PCR reaction was individually performed on the sample DNA prepared as described above, digestion was performed using a restriction enzyme that specifically recognizes the base sequence of the region containing each polymorphic site of each Cathespin H gene. Then, electrophoresis is performed on a gel, and the presence or absence of a region containing each polymorphic site of the Cathespin H gene in the sample is confirmed by the presence or absence of cleavage, and the suitability between the donor and the patient in HCT therapy is determined.

(4) Method by direct base sequence determination
PCR primers are set so as to amplify the region containing the polymorphic site of the Cathepsin H gene, and the PCR reaction is individually performed on the sample DNA prepared as described above. The PCR amplification product is used as a template after purification, and the base sequence is directly determined using the primers used for PCR, and the base sequence of the amplified portion of the region containing the polymorphic site of the Catthepsin H gene is directly read. Based on the results of the sequence, the suitability of donor and patient in HCT therapy is determined.

  In addition, diagnosis can also be made by using mass spectrometry such as Invador method, MALDI-TOF / MS method, PCR-SSCP (single strand conformation polymorphism), and RCA method. Furthermore, diagnosis can also be performed using a DNA chip in which a region containing the polymorphic site of the Cathespin H gene is fixed. These methods are publicly known, and can be performed, for example, according to the description of the post-sequence genomic chemistry 1 SNP gene polymorphism strategy edited by Kenichi Matsubara et al.

  Based on the results of the various methods using PCR as described above, the polymorphic site of Cathepsin H protein derived from donors and patients in HCT therapy and / or the nucleotide sequence encoding the mHA epitope peptide of Cathepsin H protein are confirmed. The suitability of donors and patients in Japan can be determined and diagnosed as a useful combination for immunotherapy to enhance GVT. Below, an example of the determination based on the result of having performed the genotyping between a donor and a patient about the obtained PCR amplification product is shown. However, it is not limited to this.

  Includes a useful polymorphic site of the Cathepsin H gene to confirm the compatibility of Cathepsin H protein between donors and patients with HLA-A33 or A31 and to determine if immunotherapy to enhance the patient's GVT is effective As an example of the region, the mHA epitope peptide of SEQ ID NO: 4 and / or SEQ ID NO: 5 can be used.

For example, as a result of the various PCR methods, (1) a DNA sample prepared from a donor contains a base sequence that hybridizes with the base sequence encoding the mHA epitope peptide of SEQ ID NO: 4, and (2) DNA prepared from a patient When a base sequence that hybridizes with the base sequence encoding the mHA epitope peptide of SEQ ID NO: 5 is present in the sample, both Catthepsin H proteins are incompatible, and in this case, the CD8 ⁺ CTL derived from the donor is the patient's GVT. Can strengthen.

Conversely, (1) a DNA sequence prepared from a donor has a base sequence that hybridizes with a base sequence encoding the mHA epitope peptide of SEQ ID NO: 5, and (2) in a DNA sample prepared from a patient, Even in the presence of a base sequence that hybridizes with the base sequence encoding the mHA epitope peptide of SEQ ID NO: 4, both Catthepsin H proteins are incompatible, and donor-derived CD8 ⁺ CTL can enhance the patient's GVT.

Furthermore, (1) a DNA sequence prepared from a donor contains a base sequence that hybridizes with the base sequence encoding the mHA epitope peptide of SEQ ID NO: 4 or 5 (that is, the donor is homologous to the base sequence of SEQ ID NO: 4 or 5). (2) When a base sequence that hybridizes with the base sequence encoding the mHA epitope peptides of SEQ ID NOs: 4 and 5 is present in the DNA sample prepared from the patient (ie, the patient has SEQ ID NO: 4 and Both Catthepsin H proteins are incompatible, and donor-derived CD8 ⁺ CTL can enhance the patient's GVT.

However, DNA samples prepared from donors and DNA samples prepared from patients are SEQ ID NO: 4 or 5 (ie when homozygous for the nucleotide sequence of SEQ ID NO: 4 or 5) or 4 and 5 (ie SEQ ID NO: 4 and 5). If there is a base sequence that hybridizes with the base sequence encoding the mHA epitope peptide of the same SEQ ID NO.), Both Catthepsin H proteins are compatible and are derived from the donor. CD8 ⁺ CTL is not suitable for enhancing the patient's GVT.

  Although the PCR method can be carried out by using a single nucleotide sequence encoding any of the peptide sequences of these combinations of peptides, the accuracy of the determination results can be improved and the DNA fragment can be derived. In order to reliably confirm the above, it is desirable to use a combination of the base sequences encoding these peptides.

  On the other hand, the opposite reaction to the GVT reaction is the host versus graft reaction, which is a dangerous reaction that leads to rejection of donor hematopoietic cells transfused during HCT, but with a long history of transfusion, aplastic anemia and bone marrow. The peptides of the present invention may also be used to predict the risk of transplanted hematopoietic cell rejection, which is relatively likely to occur in patients with dysplastic syndromes.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[Example 1] Identification of CD8 ⁺ CTL epitope peptide capable of damaging hematopoietic tumor cells Preparation of various materials
(1) Obtaining blood Before and after transplantation of peripheral blood and HCT in patients undergoing HCT from HLA-matched siblings for leukemia (two cases of acute myeloid leukemia (HLA-A33 and HLA-A31)), a hematopoietic tumor Mononuclear cells were isolated from donor peripheral blood. HLA type was measured using DNA typing method.

(2) EBV-infected B cell line
Peripheral blood mononuclear cells in HCT donors and patients before transplantation are infected with the supernatant of B95-8 cells (including live EBV virus), which is an EBV-producing cell line, and EBV-infected B cell lines (Lymphoblastoid cell line, below) , Referred to as EBV-infected LCL).
Similarly, EBV-infected LCL was established by infecting the peripheral blood mononuclear cells of healthy individuals with HLA-A33 or HLA-A31 with the supernatant of B95-8 cells, which are EBV-producing cell lines.

2. Establishment of culture of CD8 ⁺ CTL specific for mHA epitope peptide
(1) CD8 ⁺ CTL specific for mHA epitope peptide
The post-HCT patient peripheral blood mononuclear cells obtained in 1. (1) above were mixed and cultured in a flask together with the pre-HCT patient peripheral blood mononuclear cells that had been irradiated with a lethal dose of radiation. There was an incompatibility of the mHA epitope peptide between the donor and the patient, and CD8 ⁺ CTL derived from donor lymphocytes in response to the patient's mHA epitope peptide (antigen) started to proliferate. To help CD8 ⁺ CTL growth, IL-2 was added to the culture.

(2) Establishment of CD8 ⁺ CTL clone specific for patient's mHA epitope peptide and cytotoxicity test
2. CD8 ⁺ CTL clones were established from the two CD8 ⁺ CTL lines obtained from the two cases grown in (1) using the limiting dilution method. A plurality of CTL clones were obtained from each CD8 ⁺ CTL line, but the response patterns to EBV-infected LCL prepared from healthy individuals were equivalent, so it was considered to be a daughter clone originating from the same cell. Therefore, one CTL clone with good growth was selected from each CD8 ⁺ CTL strain and subjected to the following experiment. The two selected CTL clones did not damage the EBV-infected LCL of the HCT donor in the cytotoxicity test using the ⁵¹ Cr release method, but were specific to the patient's mHA epitope peptide because the EBV-infected LCL of the patient was damaged. It was considered the target. In addition, from the reactivity to multiple EBV-infected LCL prepared from healthy individuals with HLA-A33 or HLA-A31, two CD8 ⁺ CTL clones were presented by HLA-A33 and HLA-A31, respectively. (Hereinafter referred to as HLA-A33 restricted CD8 ⁺ CTL clone, HLA-A31 restricted CD8 ⁺ CTL clone).

Furthermore, as shown in FIG. 7, one hematopoietic tumor patient (a patient with acute myeloid leukemia FAB classification M5b type, who was established from peripheral blood after transplantation of HLA-A31-restricted CD8 ⁺ CTL clone, and HLA-A33 was also The leukemia cells at the first onset were collected and the cytotoxicity of the HLA-A33-restricted CD8 ⁺ CTL clone was examined. As a result, not only EBV-infected LCL established from the same patient but also acute myeloid leukemia cells were damaged. It was shown that hematopoietic tumor cells were actually damaged. In addition, the U937 leukemia cell line introduced with HLA-A33 and normal T cells and B cell blasts established from the same patient were not strongly damaged, suggesting that the expression of the Cathepsin H gene may be weak in these cell types. It was.

3. Identification of the gene encoding mHA by linkage analysis
(1) Preparation of cell panel and cytotoxicity test We obtained EBV-infected LCL of CEPH (Centre d'Etude du Polymorphisme Humain) family from an international tissue fit workshop, and HLA-A33 and HLA-A31 were collected in each EBV-infected LCL. Each of the genes was introduced to prepare each cell panel that expresses HLA-A33 or HLA-A31 on the cell surface. Using these HLA-A33-restricted CD8 ⁺ CTL clone and HLA-A31-restricted CD8 ⁺ CTL clone obtained in 2. (2) as a target cell, a cytotoxicity test using the ⁵¹ Cr release method was performed. We examined which EBV-infected LCL contains mHA epitope peptides with the same polymorphism as hematopoietic tumor patients. As a result, the same cytotoxicity pattern was shown in the HLA-A33-restricted CD8 ⁺ CTL clone and the HLA-A31-restricted CD8 ⁺ CTL clone. The results of HLA-A33 restricted CD8 ⁺ CTL clone and HLA-A31 restricted CD8 ⁺ CTL clone are shown in FIG.

(2) Two-point linkage analysis FASTLINK software and CEPH family gene analysis databases published on the Internet are available at ftp://fastlink.nih.gov/pub/fastlink and http://www.cephb.fr, respectively. / cephdb / than obtain and 3. results and combinations of cytotoxic tests obtained in (1), 2 (1) obtained in HLA-A33-restricted CD8 ⁺ CTL clones and HLA-A31-restricted CD8 ⁺ CTL When the locus recognized by the clone was identified, it was found to be around the long arm 24-25 of chromosome 15. Of the 34 genes at this locus, we searched for genes that have one or more polymorphic sites and could encode multiple mHA epitope peptides presented on both HLA-A33 and HLA-A31 However, there were several candidates, and the gene was not identified. Therefore, messenger RNA was isolated from EBV-infected LCL of a patient with HLA-A33 who established an HLA-A33-restricted CD8 ⁺ CTL clone, a complementary DNA library was created, and the gene was searched by expression cloning method. Cathepsin H, which is present on the long arm 24-25 of chromosome 15, was identified.

4). Identification of mHA epitope peptide capable of binding to HLA-A33 or HLA-A31 The amino acid sequence of Cathesin H protein shown in SEQ ID NO: 1 can be used to search for gene polymorphism. According to http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=2289702 of the National Center for Biotechnology Information (NCBI) of Health (NIH), polymorphisms with 3 amino acid substitutions Existence is announced. To compare the nucleotide sequences of the Cathepsin H gene in DNA obtained from multiple EBV-infected LCLs that do not damage the polymorphic sites involved in the generation of mHA epitope peptides among HLA-A33-restricted CD8 ⁺ CTL clones As a result of the search, it was found that the polymorphism of amino acid No. 11 controls the presence or absence of cytotoxicity of HLA-A33-restricted CD8 ⁺ CTL clone. The peptide sequence of this site is collated by HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html.) Of BioInformatics & Molecular Analysis Section (BIMAS), and HLA-A33 or HLA A large number of mHA epitope candidate peptides consisting of 9 to 10 amino acids having a binding motif of -A31 were screened, and four mHA epitope candidate peptides were synthesized. This was added to mHA-negative HCT donor cells that had been radiolabeled with ⁵¹ Cr and allowed to stand at room temperature for 30 minutes, and then the appropriate number of HLA-A33-restricted CD8 obtained in 2. (2). Cytotoxicity test was performed by mixing with ⁺ CTL clone or HLA-A31 restricted CD8 ⁺ CTL clone.

In addition, in order to confirm that the CD8 ⁺ CTL mHA epitope peptide is endogenously processed, a minigene encoding each polymorphic site of the Cathepsin H gene was created, and mHA-negative and restricted HLA was expressed in advance. The interferon γ release test was performed by introducing the 293T cells into the antigen-presenting cells (FIG. 9). Here, endogenous processing means that Cathesepsin H protein newly produced in a minigene-transfected cell is cleaved into an antigen peptide of the correct length by the antigen processing mechanism inherent in the cell, and the HLA heavy chain And β2 microglobulin form a complex and is displayed on the surface of the Cathepsin H minigene-introduced cell.

According to the test method described above, the following mHA epitope peptides represented by SEQ ID NOs: 4 to 7 reacted with CD8 ⁺ CTL and the CD8 ⁺ CTL clone were obtained. Among these peptides, SEQ ID NOs: 4 and 5 are peptide sequences having an HLA-A33 binding motif, and SEQ ID NOs: 4 to 7 are peptide sequences having an HLA-A31 binding motif. That is, HLA-A31 can bind and display both 9 or 10 amino acids.

The mHA epitope peptides of SEQ ID NOs: 4 to 7 are partial peptides comprising the 11th amino acid in the amino acid sequence of the Cathespin H protein represented by SEQ ID NO: 2 (SEQ ID NO: 3).
Trp Ala Thr Leu Pro Leu Leu Cys Ala Arg (SEQ ID NO: 4)
Trp Ala Thr Leu Pro Leu Leu Cys Ala Gly (SEQ ID NO: 5)
Ala Thr Leu Pro Leu Leu Cys Ala Arg (SEQ ID NO: 6)
Ala Thr Leu Pro Leu Leu Cys Ala Gly (SEQ ID NO: 7)

[Example 2] Identification of mHA epitope peptide capable of binding to HLA-A2 The peptides shown in SEQ ID NOs: 4 to 7 are partial peptides each containing the 11th amino acid of Cathespin H protein, and Although it was an mHA epitope peptide that binds to HLA-A33 or HLA-A31, the amino acid sequence of Catthepsin H protein was published on the Internet in order to further examine the mHA epitope peptide that can bind to HLA-A2. As a result of comparison with HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html.) Of BioInformatics & Molecular Analysis Section (BIMAS), 9-9 having an HLA-A2 binding motif It was found that an mHA epitope consisting of 10 amino acids was present, and the following 8 epitope candidate peptides were identified.

Among these sequences, the peptides represented by SEQ ID NOs: 8 to 15 are partial peptides containing the 11th amino acid in the amino acid sequence of Cathesin H protein.
Leu Leu Cys Ala Arg Ala Trp Leu Leu (SEQ ID NO: 8)
Leu Leu Cys Ala Arg Ala Trp Leu Leu (SEQ ID NO: 9)
Pro Leu Leu Cys Ala Arg Ala Trp Leu (SEQ ID NO: 10)
Pro Leu Leu Cys Ala Gly Ala Trp Leu (SEQ ID NO: 11)
Arg Ala Trp Leu Leu Gly Val Pro Val (SEQ ID NO: 12)
Gly Ala Trp Leu Leu Gly Val Pro Val (SEQ ID NO: 13)
Pro Leu Leu Cys Ala Arg Ala Trp Leu Leu (SEQ ID NO: 14)
Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu (SEQ ID NO: 15)

[Example 3] Vaccine injection
Each peptide of SEQ ID NOs: 4 to 17 was dissolved in DMSO to a final concentration of 20 mg / ml, and sterilized by filtration. The obtained peptide-containing solution was dispensed and sealed in 1 ml sterilized vials to obtain a vaccine injection.

It is a figure which shows the recognition mechanism of the hematopoietic organ cell or solid tumor cell by CD8 ⁺ CTL. It is a figure which shows the preparation method of MHC-tetramer. It is a figure which shows the coupling | bonding of MHC-tetramer and CD8 ⁺ CTL. It is a figure which shows the preparation method of MHC-magnetic bead. It is a figure which shows isolation of CD8 ⁺ CTL by MHC-magnetic bead. It is a figure which shows the proliferation of CD8 ⁺ CTL by the stimulation by mHA epitope peptide and subsequent anti-CD3 antibody and IL-2. It is a figure which shows the cytotoxicity with respect to various target cells including the leukemia cell line by CD8 ⁺ CTL couple | bonded with a HLA-A24 restriction | limiting clone. HLA-A33-restricted clones, HLA-A31-restricted clones expressed HLA-A33 and HLA-A31 in EBV-infected LCL established from the CEPH family (family numbers 1347 and 1413), It is a figure which shows having injured with the completely same pattern. It is a figure which shows the result of the identification of the epitope peptide which each CTL clone recognizes.

Claims (27)

A minor histocompatibility antigen (mHA) epitope peptide that is a partial peptide of Cathepsin H protein and can be recognized by CD8 ⁺ cytotoxic T lymphocytes in combination with human major histocompatibility antigen (HLA).   The minor histocompatibility antigen (mHA) epitope peptide according to claim 1, wherein the partial peptide of the Cathepsin H protein has 5 to 20 amino acid residues.   The minor histocompatibility antigen (mHA) epitope peptide according to claim 2, wherein the partial peptide of the Cathepsin H protein has 9 to 10 amino acid residues.   The peptide according to any one of claims 1 to 3, wherein the partial peptide of the Cathepsin H protein is a partial peptide comprising the 11th amino acid in the amino acid sequence represented by SEQ ID NO: 1.   The peptide according to any one of claims 1 to 4, wherein the HLA is HLA-A33.   The peptide of Claim 5 which is a peptide which consists of an amino acid sequence shown by sequence number 3 or 4.   The peptide according to any one of claims 1 to 4, wherein the HLA is HLA-A31.   The peptide of Claim 7 which is a peptide selected from the group which consists of an amino acid sequence shown by sequence number 4-7.   The peptide according to any one of claims 1 to 4, wherein the HLA is HLA-A2.   The peptide of Claim 9 which is a peptide selected from the group which consists of an amino acid sequence shown by sequence number 8-15. The peptide according to any one of claims 1 to 10, wherein the CD8 ⁺ cytotoxic T lymphocytes damage hematopoietic tumor cells or solid tumor cells.   The peptide according to claim 11, wherein the hematopoietic tumor is leukemia, myelodysplastic syndrome, malignant lymphoma or multiple myeloma.   The peptide according to claim 11, wherein the solid tumor is renal cell carcinoma, malignant melanoma, breast cancer, gastric cancer, lung cancer or prostate cancer.   A hematopoietic tumor or solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation comprising the peptide according to any one of claims 1 to 13 or an antigen-presenting cell pulsed with the peptide as an active ingredient, or Vaccine to prevent recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation. After transplantation of allogeneic hematopoietic cells, comprising CD8 ⁺ cytotoxic T lymphocytes obtained by stimulating peripheral blood lymphocytes with the peptide according to any one of claims 1 to 13 or antigen-presenting cells pulsed with the peptide. A passive immunotherapeutic agent for treating residual or recurrent hematopoietic tumor or solid tumor, or preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation. A major histocompatibility antigen complex and / or a major histocompatibility antigen complex-tetramer prepared from the peptide according to any one of claims 1 to 13 is reacted with peripheral blood lymphocytes to produce the major histocompatibility antigen complex. body and / or major histocompatibility complex - tetramer CD8 ⁺ cytotoxic T lymphocytes to form a conjugate bound, including CD8 ⁺ cytotoxic T lymphocytes obtained by isolating from the conjugate, A passive immunotherapeutic agent for treating hematopoietic tumor or solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation or preventing recurrence of hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation. A major histocompatibility complex-labeled magnetic bead prepared from the peptide according to any one of claims 1 to 13 is reacted with peripheral blood lymphocytes, and the major histocompatibility complex-labeled magnetic bead is subjected to CD8 ⁺ A hematopoietic tumor or solid tumor that has remained or recurred after allogeneic hematopoietic cell transplantation, comprising a CD8 ⁺ cytotoxic T lymphocyte obtained by forming a conjugate in which cytotoxic T lymphocytes are bound and isolated from the conjugate Or a passive immunotherapeutic agent for preventing recurrence of a hematopoietic tumor or solid tumor after allogeneic hematopoietic cell transplantation. Peripheral blood is stimulated with the peptide according to any one of claims 1 to 13 to obtain CD8 ⁺ cytotoxic T lymphocytes, and cytokines and / or chemokines produced by the CD8 ⁺ cytotoxic T lymphocytes are obtained. A method for quantifying CD8 ⁺ cytotoxic T lymphocytes that damage hematopoietic tumor cells or solid tumor cells, comprising measuring. A major histocompatibility complex and / or major histocompatibility complex and / or major histocompatibility is prepared from the peptide according to claim 1. A method for quantifying CD8 ⁺ cytotoxic T lymphocytes that damages hematopoietic tumor cells or solid tumor cells in the peripheral blood, wherein the complex-tetramer is reacted with peripheral blood.   A method for diagnosis of compatibility with allogeneic hematopoietic cell transplantation, comprising genotyping DNA derived from donors and patients in allogeneic hematopoietic cell transplantation using the base sequence of the region containing the polymorphic site of the Catthepsin H gene shown in SEQ ID NO: 2.   DNA from donors and patients in allogeneic hematopoietic cell transplantation is amplified by the PCR method using the base sequence of the region containing the polymorphic site of the Catepsin H gene shown in SEQ ID NO: 2, and the obtained PCR amplification product is between the donor and the patient. 21. The allogeneic hematopoietic cell transplantation compatibility diagnostic method according to claim 20, which comprises performing genotyping.   The allogeneic hematopoietic cell transplantation compatibility diagnostic method according to claim 20 or 21, wherein the base sequence of the region containing the polymorphic site of the Cathepsin H gene has a base length of 18 to 30 mer.   The base sequence of the region containing the polymorphic site of the Catthepsin H gene shown in SEQ ID NO: 1 is the base sequence encoding the mHA epitope peptide according to claim 1, Diagnostic method.   An isolated antigen-presenting cell pulsed with at least one peptide of any one of claims 1 to 13, wherein the peptide is bound to HLA expressed on the surface. A method for activating an antigen-presenting cell, comprising culturing an antigen-presenting cell in vitro with at least one peptide of the peptide according to any one of claims 1 to 13, wherein the activated antigen An activation method in which presenting cells can stimulate CD8 ⁺ cytotoxic T lymphocytes. A method for preparing CD8 ⁺ cytotoxic T lymphocytes that can be used for the prevention and treatment of hematopoietic tumors or solid tumors by any of the following methods (a) to (c).
(a) a method comprising stimulating peripheral blood lymphocytes in vitro using the peptide according to any one of claims 1 to 13 or an antigen-presenting cell pulsed with the peptide,
(b) reacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the peptide according to any one of claims 1 to 13 with peripheral blood lymphocytes; histocompatibility complex and / or major histocompatibility complex - tetramer CD8 ⁺ cytotoxic T lymphocytes to form a conjugate bound to isolate CD8 ⁺ cytotoxic T lymphocytes from conjugate A method comprising: and
(c) The main histocompatibility complex-labeled magnetic beads prepared from the peptide according to any one of claims 1 to 13 are reacted with peripheral blood lymphocytes, and the main histocompatibility complex-labeled magnetic beads are reacted. Forming a conjugate with CD8 ⁺ cytotoxic T lymphocytes bound thereto, and isolating CD8 ⁺ cytotoxic T lymphocytes from the conjugate 27. Isolated CD8 ⁺ cytotoxic T lymphocytes obtained by the method of claim 26, which can be used for the prevention and treatment of hematopoietic or solid tumors.

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