JP2004099509A - CD8+CYTOTOXIC LYMPHOCYTE mHA EPITOPIC PEPTIDE AND APPLICATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CD8<SP>+</SP>cytotoxic T lymphocyte epitopic peptide useful for treating hematopoietic organ tumor or solid tumor remaining or relapsed after homoplastic hematopoietic cell transplantation or preventing the relapse of hematopoietic organ tumor or solid tumor after homoplastic hematopoietic cell transplantation. <P>SOLUTION: The CD8<SP>+</SP>cytotoxic T lymphocyte epitopic peptide as a minor histocompatible antigen(mHA) epitopic peptide is a partial peptide consisting of 9-10 amino acid residues of bcl2A1 protein and recognizable by CD8<SP>+</SP>cytotoxic T lymphocyte through binding to a human leukocyte antigen(HLA). <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】ＣＤ８^＋ＣＴＬによる造血器腫瘍細胞又は固形腫瘍細胞の認識機構を示す図である。
【図２】ＭＨＣ−テトラマーの調製方法を示す図である。
【図３】ＭＨＣ−テトラマーとＣＤ８^＋ＣＴＬの結合を示す図である。
【図４】ＭＨＣ−磁気ビーズの調製方法を示す図である。
【図５】ＭＨＣ−磁気ビーズによるＣＤ８^＋ＣＴＬの単離を示す図である。
【図６】ｍＨＡエピトープペプチド、及びその後の抗ＣＤ３抗体、ＩＬ−２による刺激による、ＣＤ８^＋ＣＴＬの増殖を示す図である。
【図７】ＨＬＡ−Ａ２４拘束性クローンと結合するＣＤ８^＋ＣＴＬによる種々の白血病細胞の細胞傷害性を示す図である。
【図８】ＨＬＡ−Ａ２４拘束性クローン、ＨＬＡ−Ｂ４４拘束性クローンが、ＣＥＰＨ家系より樹立したＥＢＶ感染ＬＣＬに各々ＨＬＡ−Ａ２４、ＨＬＡ−Ｂ４４を発現させたものに対して、全く同様なパターンで傷害するのを示す図である。
【図９】本発明の配列番号４のペプチドにより調製したテトラマーによる、造血器腫瘍細胞又は固形腫瘍細胞を傷害するＣＤ８^＋ＣＴＬの同定と定量を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a partial peptide comprising 9 to 10 amino acid residues of the bcl2A1 protein, and which binds to human major histocompatibility antigen (HLA) to form CD8. ⁺ A minor histocompatibility antigen (mHA) epitope peptide that can be recognized by cytotoxic T lymphocytes (hereinafter referred to as CTL), a hematopoietic tumor that has remained or recurred after allogeneic hematopoietic cell transplantation using the peptide, or Vaccines for treating solid tumors or preventing recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation and passive immunotherapy, CD8 damaging hematopoietic tumor cells or solid tumor cells ⁺ The present invention relates to a method for quantifying cytotoxic T lymphocytes and a method for diagnosing suitability for allogeneic hematopoietic cell transplantation using the peptide.
[0002]
[Prior art]
Allogeneic hematopoietic cell transplantation (hereinafter, referred to as HCT) has been established as a treatment method that can cure hematopoietic tumors such as leukemia and the like whose prognosis is poor with chemotherapy alone. More recently, HCT called mini-transplantation has also been attempted for solid tumors, and some solid tumors (eg, renal cell carcinoma, malignant melanoma, etc.) are beginning to show a tumor shrinking effect. In fact, some solid tumors have been reported to have abnormally expressed genes specific to hematopoietic cells. For example, the bcl2A1 gene is abnormally expressed in gastric cancer cells and malignant melanoma, 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 performing HCT, and the prognosis is not always good.
[0003]
In HCT, hematopoietic stem cells are provided to a patient from a hematopoietic stem cell donor (hereinafter, referred to as a donor) to which a human major histocompatibility antigen (hereinafter, referred to as HLA) is compatible, and the HLA is identical between the donor and the patient. However, many mismatches occur in minor histocompatibility antigens (hereinafter, referred to as mHA) derived from various gene polymorphisms, and the target is mainly targeted to donor-derived T lymphocytes. An immune response occurs within the patient. If this immune response exerts an antitumor effect on the patient's hematopoietic or solid tumor, it will lead to cure of the underlying disease and avoid recurrence. This is called a graft-versus-tumor (hereinafter referred to as GVT) effect.
[0004]
In patients undergoing recurrence after HCT, the GVT effect is considered to be insufficient.
For this reason, in order to enhance the action of donor-derived T lymphocytes and to enhance the GVT effect, treatment may be performed to reduce the amount of immunosuppressants used for rejection prevention. In this case, donor-derived T lymphocytes are used. And strongly exclude cells in the patient's body as non-self, often resulting in a fatal complication called graft-versus-host disease (GVHD). For this reason, there is a demand for an effective treatment that avoids GVHD while actively extracting and utilizing only the GVT effect.
[0005]
The main immunocompetent cells controlling the recurrence of hematopoietic or solid tumors after HCT are CD8 from donor T lymphocytes ⁺ CTL. CD8 from donor ⁺ CTLs have the ability to destroy remaining hematopoietic tumor cells or solid tumor cells when they are found in a patient's body, recognizing them as non-self. Therefore, CD8 ⁺ Effective activation of the CTL's injury function would enhance the GVT effect and avoid the onset of GVHD, leading to the development of new hematopoietic or solid tumor recurrence prevention and treatment methods. Probability is high.
[0006]
CD8 ⁺ When CTL recognizes hematopoietic tumor cells or solid tumor cells, it has the following features.
(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 hematopoietic tumor cells or solid tumor cells of a patient encoded by a gene having a genetic polymorphism unique to an individual. For example, a peptide consisting of 9 to 10 amino acids encoded by a gene having an individual-specific polymorphism in the bcl2A1 protein, which differs between the donor and the patient in the bcl2A1 protein. Damages (destroys) solid tumor cells.
(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 differs between races and individuals, and different HLAs may result in different peptides serving as mHA epitopes, so that presentation to CTL itself may not be possible.
[0007]
As is clear from the above (1) to (4), CD8 that damages hematopoietic tumor cells or solid tumor cells ⁺ Identifying an mHA protein comprising a peptide that can be a CTL mHA epitope peptide and determining the amino acid sequence of the epitope peptide without excess or deficiency can result in an effective GVT effect in a patient whose donor has undergone or has undergone HCT. It can be an essential confirmation when diagnosing or not and applying specific immunotherapy.
[0008]
However, the 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 possess HLA-B44, 24%; HLA-B52, 20%; HLA-B35, 15%; HLA-B54, 14%; HLA-Cw1, 34%; HLA-Cw * 14 HLA-Cw * 0702, 22%; HLA-Cw * 0801, 21%; HLA-Cw * 1202, 21%, etc.), especially HLA-A24. , HLA-B44 type, etc., CD8 ⁺ There are very few reports of mHA epitope peptides recognized by CTLs, and mHA useful for treating residual or recurrent hematopoietic tumors or solid tumors after HCT, or preventing recurrence of hematopoietic tumors or solid tumors after HCT. There is a need for the development of epitope peptides.
[0009]
[Non-patent document 1]
Choi et al., Oncogene, Vol. 11, No. 9, pp. 1693-8, 1995
[Non-patent document 2]
Kenny et al., Oncogene, Vol. 14, No. 8, pp. 997-1001, 1997
[Non-Patent Document 3]
Fujii et al., Transplantation Vol. 73, No. 7, pp. 1371-1141, 2002
[0010]
[Problems to be solved by the invention]
The present invention relates to a partial peptide comprising 9 to 10 amino acid residues of the bcl2A1 protein, and which binds to HLA to form CD8. ⁺ MHA epitope peptide recognizable by CTLs, treating hematopoietic tumors or solid tumors remaining or recurring after allogeneic hematopoietic cell transplantation using the peptide, or treating hematopoietic tumors or solid tumor relapse after allogeneic hematopoietic cell transplantation Vaccine for prevention and passive immunotherapy, CD8 damaging hematopoietic tumor cells or solid tumor cells ⁺ An object of the present invention is to provide a method for quantifying CTLs and a method for diagnosing suitability for allogeneic hematopoietic cell transplantation using the peptide.
[0011]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, CD8 that damages hematopoietic tumor cells or solid tumor cells ⁺ The present inventors have found an mHA epitope peptide that can be recognized by CTL, and have completed the present invention.
[0012]
That is, the present invention provides the following (1) to (19).
(1) A partial peptide consisting of 9 to 10 amino acid residues of the bcl2A1 protein, which binds to human major histocompatibility antigen (HLA) to form CD8 ⁺ A minor histocompatibility antigen (mHA) epitope peptide that can be recognized by cytotoxic T lymphocytes.
(2) A partial peptide comprising 9 to 10 amino acid residues in the bcl2A1 protein, wherein the partial peptide comprises the 19th, 39th or 82nd amino acids in the amino acid sequence represented by SEQ ID NO: 1. The peptide according to (1), which is:
(3) The peptide according to (1) or (2), wherein the HLA is HLA-A24.
(4) The peptide according to (3), which is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4.
(5) The peptide according to (1) or (2), wherein the HLA is HLA-B44.
[0013]
(6) The peptide according to (5), which is a peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 5 to 8.
(7) The peptide according to (1) or (2), wherein the HLA is HLA-A2.
(8) The peptide according to (7), which is a peptide selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 9 to 16.
(9) CD8 ⁺ The peptide according to any one of (1) to (8), wherein the cytotoxic T lymphocyte damages hematopoietic tumor cells or solid tumor cells.
(10) The peptide according to (9), wherein the hematopoietic organ tumor is leukemia, myelodysplastic syndrome, malignant lymphoma, or multiple myeloma.
[0014]
(11) The peptide according to (9), wherein the solid tumor is renal cell carcinoma, malignant melanoma, breast cancer, stomach cancer, lung cancer or prostate cancer.
(12) Treating a hematopoietic tumor or a solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation, comprising as an active ingredient the peptide according to any of (1) to (11) or an antigen-presenting cell pulsed with the peptide. A vaccine for preventing recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation.
(13) CD8 obtained by stimulating peripheral blood lymphocytes with the peptide according to any of (1) to (11) or antigen-presenting cells pulsed with the peptide. ⁺ Passive treatment to treat residual or recurrent hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation, including cytotoxic T lymphocytes, or to prevent recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation Immunotherapy.
(14) reacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the peptide according to any one of (1) to (11) with peripheral blood lymphocytes, CD8 to compatible antigen complex and / or major histocompatibility complex-tetramer ⁺ CD8 obtained by forming a conjugate to which cytotoxic T lymphocytes are bound and isolating from the conjugate ⁺ Passive treatment to treat residual or recurrent hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation, including cytotoxic T lymphocytes, or to prevent recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation Immunotherapy.
(15) The major histocompatibility complex-labeled magnetic beads are reacted with the major histocompatibility complex-labeled magnetic beads prepared from the peptide according to any of (1) to (11) and peripheral blood lymphocytes. CD8 ⁺ CD8 obtained by forming a conjugate to which cytotoxic T lymphocytes are bound and isolating from the conjugate ⁺ Passive treatment to treat residual or recurrent hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation, including cytotoxic T lymphocytes, or to prevent recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation Immunotherapy.
[0015]
(16) CD8 by stimulating peripheral blood with the peptide according to any of (1) to (11) ⁺ Obtaining a cytotoxic T lymphocyte, wherein said CD8 ⁺ CD8 that damages hematopoietic tumor cells or solid tumor cells, characterized by measuring cytokines and / or chemokines produced by cytotoxic T lymphocytes ⁺ A method for quantifying cytotoxic T lymphocytes.
(17) A major histocompatibility complex and / or a major histocompatibility complex-tetramer is prepared from the peptide according to any of (1) to (11), and the major histocompatibility complex and / or the major histocompatibility complex is obtained. CD8 that causes hematopoietic tumor cells or solid tumor cells in peripheral blood to react with histocompatibility complex-tetramer and peripheral blood ⁺ A method for quantifying cytotoxic T lymphocytes.
(18) DNA from donors and patients in allogeneic hematopoietic cell transplantation is amplified by PCR using the nucleotide sequence of the region containing the polymorphic site of the bcl2A1 gene shown in SEQ ID NO: 2, and the resulting PCR amplification product A method for diagnosing suitability for allogeneic hematopoietic cell transplantation, comprising performing genotyping between patients.
(19) The diagnostic method according to (18), wherein the nucleotide sequence of the region including the polymorphic site of the bcl2A1 gene shown in SEQ ID NO: 2 is a nucleotide sequence encoding the mHA epitope peptide according to (1).
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in more detail.
1. CD8 ⁺ MHA epitope peptide that can be recognized by CTL
The peptide in the present invention means a linear amino acid molecular chain which has physiological activity and is mutually connected by a peptide bond between an α-amino group and a carboxyl group of adjacent amino acid residues. Peptides are not meant to be of a particular length, but may be of various lengths. It may be in an uncharged or salt form, and in some cases, may be modified by glycosylation, amidation, phosphorylation, carboxylation, phosphorylation, or the like. Furthermore, one or several (for example, 1 to 8, preferably 1 to 8) epitopes of the present invention do not substantially alter the physiological and immunological activities of the epitope of the present invention as long as they do not have a harmful activity when administered. Peptides in which insertion, addition, substitution, etc. of (.about.3) amino acids have occurred are also included in the present invention. For example, those in which an additional amino acid sequence is interposed at the N-terminus or C-terminus of the peptide are also included. Further, the peptide of the present invention can be used in the form of a complex to which a saccharide, polyethylene glycol, lipid, or the like is added, a derivative with a radioisotope, or a polymer.
[0017]
Also, the CD8 according to the present invention ⁺ An mHA epitope peptide that can be recognized by CTLs is defined as binding to HLA on the surface of hematopoietic tumor cells or solid tumor cells and expressing CD8 from the donor. ⁺ CD8 by binding to T cell antigen receptor (TCR) of CTL ⁺ A partial peptide of mHA protein present in hematopoietic tumor cells or solid tumor cells that can be recognized by CTLs, including a polymorphic site encoded by a gene having a genetic polymorphism unique to an individual. Means a peptide consisting of two amino acids. Thus, the peptide can directly injure (destruct) and eliminate certain types of hematopoietic or solid tumor cells of HLA that express the mHA protein, CD8 ⁺ An antigen group that activates the CTL's cellular immune system. CD8 ⁺ CTL is CD8 expressing CD8 which is one of surface antigen molecules present on human lymphocytes. ⁺ Means cytotoxic T lymphocytes. FIG. 1 shows the mechanism.
[0018]
In the present invention, hematopoietic tumors include tumors occurring in hematopoietic tissues or cells such as leukemia, myelodysplastic syndrome, malignant lymphoma, and multiple myeloma. In the present invention, the solid tumor includes any solid tumor that abnormally expresses the bcl2A1 gene. Specifically, renal cell carcinoma, malignant melanoma, breast cancer, gastric cancer, lung cancer, prostate cancer And the like.
[0019]
Potential candidates for mHA proteins are various proteins that exist only in hematopoietic tumor cells or solid tumor cells and are encoded by genes having genetic polymorphisms specific to individuals. Among them, one gene has as many as three polymorphic sites and the gene is expressed only in hematopoietic-related cells and some solid tumors. Therefore, the bcl2A1 protein is one of the mHA epitope peptides of the present invention. Particularly useful for identification.
[0020]
The bcl2A1 protein is strongly induced when hematopoietic cells are activated by cytokines such as Tissue Necrosis Factor, and has a function of preventing these cells from dying following a suicide process called apoptosis. It was initially identified as a gene similar to the bcl2 gene overexpressed in lymphomas (Lin et al., Journal of Immunology, 151: 4, 1979-1988, 1993). Hematopoietic cells are destined to die in a relatively short period of time due to turnover, but the cells are kept alive until the cells are completely differentiated (Lin et al., Blood, 87: 983-992). , 1996), the bcl2A1 protein protects cells to prevent them from being activated more than necessary and killed by Tissue Necrosis Factor released during tissue inflammation (Karsan et al., Journal of Biological Chemistry, No. 271). Vol. 44, pp. 27201-27204, 1996). From these functions, it is supposed that an environment in which cells proliferate is created in hematopoietic tumors in which bcl2A1 protein is overexpressed. A similar mechanism can be considered for solid tumors (Choi et al., Oncogene, Vol. 11, No. 9, pp. 1693-8, 1995).
[0021]
The nucleotide sequence of the gene encoding the bcl2A1 protein (SEQ ID NO: 2) and the amino acid sequence of the protein (SEQ ID NO: 1) are available at http: // www. ncbi. nlm. nih. gov / entrez / viewer. fcgi? val = U29680.1.
[0022]
Regarding each of the 19th and 39th polymorphisms (SEQ ID NO: 1) in the amino acid sequence of the bcl2A1 protein, refer to the National Institute of Biotechnology Information (NCBI) http: // National Institute of Health (NIH). / Www. ncbi. nlm. nih. gov / SNP / snp_ref. cgi? This can be confirmed by type = rs & rs = 1138357.
[0023]
In addition, the 82nd polymorphism (SEQ ID NO: 1) of the amino acid sequence of the bcl2A1 protein was determined by the HLA-B44-restricted CD8 established from the patient as described in Example 1 below. ⁺ When the bcl2A1 gene was identified using CTL, CD8 against 10 target cells expressing HLA-B44 ⁺ When the presence or absence of CTL cytotoxic activity was confirmed and the comparison with the bcl2A1 entire gene nucleotide sequence was examined, it was found that a polymorphic site completely matching the cytotoxic pattern was newly found at the 82nd position. This was the first thing the present inventors have confirmed.
[0024]
Therefore, the mHA epitope peptide of the present invention is a partial peptide comprising the 19th, 39th or 82nd amino acids in the amino acid sequence represented by SEQ ID NO: 1.
[0025]
In addition, a gene polymorphism unique to an individual refers to DNA obtained from a large number of individuals (for example, 50 individuals), which is subjected to base sequence determination for the bcl2A1 gene, and examined whether there is a sequence difference between individuals. If there is a sequence difference between each individual, determine if it is a universal one found in multiple individuals and search by estimating what percentage of the population has each polymorphism Can be an individual-specific genetic polymorphism.
[0026]
Since the preferred mHA epitope peptide of the present invention differs depending on the type of HLA, it can be identified by screening a peptide that can bind in each type of HLA. Specifically, in the amino acid sequence of the bcl2A1 protein, for a partial peptide comprising the 19th, 39th or 82nd amino acids, each type of HLA (eg, HLA-A24, B44, A2, etc.) A collation medium capable of retrieving an epitope peptide consisting of 9 to 10 amino acids having a binding motif, for example, HLA Peptide Binding Predictions (/http://www.bioinformatics & molecular analysis section (BIMAS) published on the Internet: .Dcrt.nih.gov / molbio / hla_bind / index.html), a peptide that can be an mHA epitope peptide (hereinafter referred to as mHA epitope) Referred flop candidate peptide) can be screened.
[0027]
The mHA epitope candidate peptide can be obtained by ordinary chemical synthesis. For example, an organic chemical synthesis method such as a solid phase peptide synthesis method, or a DNA encoding a peptide can be prepared and prepared using recombinant DNA technology. Further, synthesis using a commercially available chemical synthesizer (for example, a peptide synthesizer manufactured by Applied Biosystems) is also possible.
[0028]
For the obtained mHA epitope candidate peptide, for example, CD8 is obtained by performing any of the following methods. ⁺ It can be determined whether the peptide is an mHA epitope peptide that can be recognized by CTL.
[0029]
(1) Method 1 for determining mHA epitope peptide
2 × 10 5 in RPMI 1640 medium containing 10% human serum ⁶ At a cell concentration of 1 ml / ml, lymphocytes isolated from an adult whose bcl2A1 gene polymorphism has been previously searched are suspended, and the lymphocytes are dendritic cells (1 × 10 5) previously separated and cultured from the same person. ⁵ / Ml) or activated B cells (1 × 10 ⁶ / Ml), and any one of the mHA epitope candidate peptides is added thereto at a concentration of 1 μg / ml. Incubate at 37 ° C. for 10 days in a carbon dioxide thermostat. On the 10th day, IL-2 was added, and thereafter, stimulation with the candidate peptide and IL-2 was repeated once a week to obtain a CD8 specific to the bcl2A1 protein polymorphism site. ⁺ Induce CTL. CD8 induced in this way ⁺ Whether or not the CTL stimulates the mHA epitope candidate peptide is determined by Elispot assay or the like. Elispot assays are reported in Miyahira Y, et al., Journal of Immunological Methods, 181: 45-54, 1995 and the like. CD8 induced by judgment ⁺ Candidate peptides stimulated by CTL are determined as the mHA epitope peptide of the present invention.
[0030]
(2) Method 2 for determining mHA epitope peptide
In the amino acid sequence of the bcl2A1 protein, a peptide library of a partial peptide consisting of 9 to 10 amino acids including the 19th, 39th or 82nd amino acids is synthesized by a conventional method. The synthesized peptide was analyzed for CD8 by ELISPOT method or intracellular IFNγ producing cell ⁺ An mHA epitope peptide to which CTL reacts is selected as the epitope peptide of the present invention.
[0031]
The peptide having the amino acid sequence of SEQ ID NOs: 3 to 16 of the present invention has been confirmed as an mHA epitope peptide as a result of the screening and determination methods as described above. The mHA epitope peptides shown in SEQ ID NOs: 3 to 16 of the present invention can be prepared by various conventional peptide synthesis methods.
[0032]
2. vaccine
The mHA epitope peptide of the present invention can be used as a vaccine in active immunopeptide vaccine therapy. That is, by administering a vaccine comprising the mHA epitope peptide of the present invention to a patient after HCT, CD8 recognizing the peptide can be obtained. ⁺ CTLs can grow in the body and help prevent and treat hematopoietic or solid tumors. As the mHA epitope peptide to be used, one or two or more peptides corresponding to the type of HLA and mHA of a patient may be used, or they may be used in combination and mixed. For example, when there are a plurality of HLAs that can present the mHA epitope peptide, two or more peptides can be used in combination. Further, for example, when two or more different mHA epitope peptides (a partial peptide containing the 19th amino acid and a partial peptide containing the 39th amino acid, etc.) are present as peptides that bind to a specific type of HLA, Two or more peptides can be used in combination.
[0033]
In addition, vaccines containing antigen-presenting cells (eg, dendritic cells, B cells, macrophages, etc.) pulsed with the mHA epitope peptide of the present invention (hereinafter referred to as mHA epitope peptide pulsed cells) can also be used. . Here, the antigen-presenting cell refers to CD8 among cells expressing on its surface HLA to which the peptide can bind. ⁺ The term "pulse" means a substance having CTL stimulating ability, and "pulsing" means, for example, mixing antigen-presenting cells and the peptide in an appropriate culture solution for about 30 minutes to 1 hour.
[0034]
A vaccine comprising the mHA epitope peptide or mHA epitope peptide pulsed cells of the present invention can be prepared using methods known in the art.
For example, such vaccines include injections or solid preparations containing the mHA epitope peptide of the present invention or mHA epitope peptide pulsed cells 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, and organic acids such as acetic acid and tartaric acid. . The mHA epitope peptide or mHA epitope peptide-pulsed cells of the present invention are pharmaceutically acceptable and excipients compatible with the activity of the peptide or the cells, such as water, saline, dextrose, ethanol, and glycerol. , DMSO, and other adjuvants, or a combination thereof. Further, if necessary, auxiliary agents such as albumin, a wetting agent, and an emulsifier may be added.
[0035]
The vaccine of the present invention can be administered by parenteral administration or oral administration, but parenteral administration is generally preferred. Parenteral administration includes injections such as subcutaneous injection, intramuscular injection, and intravenous injection, and suppositories. For oral administration, it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, and cellulose.
[0036]
The vaccines of the present invention are administered in a therapeutically effective amount. The amount 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. Usually, an appropriate dose is 1-100 mg for mHA epitope peptide and 10 for mHA epitope peptide pulsed cells per patient. ⁶ -10 ⁹ And the content of each. Further, the administration interval can be set depending on the subject and the purpose.
[0037]
3. Passive immunotherapy
The mHA epitope peptide of the present invention can be used for preparing a prophylactic or therapeutic passive immunotherapeutic agent against hematopoietic tumor recurrence after HCT. That is, 1) CD8 obtained by stimulating peripheral blood lymphocytes with the peptide. ⁺ CTL, 2) reacting peripheral blood lymphocytes with a major histocompatibility complex (hereinafter referred to as MHC) prepared from the peptide or MHC-tetramer, and converting MHC or MHC-tetramer to CD8 ⁺ CD8 obtained by forming a conjugate to which CTL is bound and isolating from the conjugate ⁺ CTL, or 3) reacting peripheral blood lymphocytes with MHC-tetramer-labeled magnetic beads prepared from the peptide, and adding CD8 to MHC-tetramer-labeled magnetic beads. ⁺ CD8 obtained by forming a conjugate to which CTL is bound and isolating from the conjugate ⁺ A passive immunotherapeutic agent comprising CTL can be obtained. MHC and MHC-tetramer using the mHA epitope peptide 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 mHA epitope peptide of the present invention, is formed in a buffer. A biotin-binding site is added in advance to the C-terminal of the recombinant HLA heavy chain protein, and biotin is added to this site after MHC formation. An MHC-tetramer is prepared by mixing a commercially available labeled dye streptavidin and biotinylated MHC at a molar ratio of 1: 4. FIGS. 2 and 3 show the obtained MHC-tetramer and its reaction mechanism, respectively. In each step, protein purification by gel filtration is preferably performed.
[0038]
CD8 capable of recognizing the mHA epitope peptide of the present invention contained in a passive immunotherapy agent ⁺ CTL can be obtained by the following preparation method.
(1) CD8 ⁺ CTL preparation method 1
Lymphocytes are separated from the peripheral blood of a patient or HCT donor after HCT, and reacted with an appropriate concentration of MHC or MHC-tetramer prepared as described above at 37 ° C. for 15 minutes. CD8 bound to mHA epitope peptide in MHC or MHC-tetramer ⁺ Since CTL is stained with a labeling dye, CD8 stained using a flow cytometer or the like is used. ⁺ Only CTL is isolated. Alternatively, it can be reacted with MHC and / or MHC-tetramer previously immobilized on a sterile plate or the like. CD8 bound to mHA epitope peptide in MHC and / or MHC-tetramer immobilized on plate ⁺ In order to isolate CTL, the CD8 remaining on the plate after washing away other unbound cells ⁺ Only the CTL is suspended in a fresh culture. CD8 thus isolated ⁺ Since CTLs can damage hematopoietic tumor cells or solid tumor cells, they are stimulated and proliferated with a T cell stimulating agent such as an anti-CD3 antibody, PHA, or IL-2 to secure the number of cells required for passive immunotherapy (FIG. 6). See).
[0039]
(2) CD8 ⁺ CTL preparation method 2
The biotinylated MHC prepared as described above is bound to streptavidin-labeled magnetic beads to prepare a conjugate (hereinafter, referred to as MHC-magnetic beads). MHC-magnetic beads are shown in FIG. Lymphocytes are separated from peripheral blood of a patient after HCT or an HCT donor, and reacted with an appropriate concentration of the MHC-magnetic beads at a lymphocyte: beads ratio of 1:10. MHC-CD8 bound to MHC / mHA epitope peptide in magnetic beads ⁺ CD8 bound to CTL ⁺ When the test tube containing CTL is placed in a magnetic field, the CD8 bound to the beads ⁺ The CTL is moved to the inner wall of the test tube on the side where the magnet is located. The mechanism is shown in FIG. Thereafter, after the other cells were washed away, the test tube was removed from the magnetic field, and the antigen-specific CD8 remaining on the inner wall of the test tube was removed. ⁺ Only the CTL is suspended in a fresh culture. CD8 thus isolated ⁺ Since CTLs can damage hematopoietic tumor cells or solid tumor cells, they are stimulated and proliferated with a T cell stimulating agent such as an anti-CD3 antibody, PHA, or IL-2 to secure the number of cells required for passive immunotherapy (FIG. 6). See).
[0040]
(3) CD8 ⁺ CTL preparation method 3
Lymphocytes isolated from peripheral blood of a patient after HCT or HCT donor are directly stimulated with the mHA epitope peptide of the present invention, or antigen-presenting cells pulsed with the peptide (eg, dendritic cells, B cells, macrophages, etc.) Stimulate with. CD8 induced by stimulation ⁺ The CTL is cultured at 37 ° C. for 7 to 10 days in a carbon dioxide thermostat. Thereafter, IL-2 was added thereto, and stimulation with the mHA epitope peptide and IL-2 or the antigen-presenting cells and IL-2 was repeated once a week to obtain the CD8 of the number of cells required for passive immunotherapy. ⁺ Secure CTL (see FIG. 6).
[0041]
CD8 obtained as described above ⁺ Since CTLs can damage hematopoietic tumor cells or solid tumor cells, they are suspended in human albumin-containing PBS or the like, and used as a prophylactic or therapeutic passive immunotherapy agent against recurrence of hematopoietic tumors or solid tumors after HCT. can do.
[0042]
4. CD8 ⁺ Quantitation of CTL
CD8 capable of recognizing the mHA epitope peptide of the present invention ⁺ Patients with high risk of recurrence of hematopoietic tumors or solid tumors after HCT (patients with high malignancy of hematopoietic tumors or solid tumors that are the primary disease and receiving strong immunosuppressive drugs after HCT) Knowing whether or not it is present in peripheral blood after HCT is important information in patient management after HCT, including determination of an appropriate amount of immunosuppressant used and prediction of risk of recurrence. CD8 that recognizes the mHA epitope peptide of the present invention and can damage hematopoietic tumor cells or solid tumor cells ⁺ Quantification of CTL can be performed by the following two methods using the mHA epitope peptide of the present invention.
[0043]
The first quantification method is to induce CD8 induced by stimulating lymphocytes isolated from peripheral blood after transplantation of HCT patients with the mHA epitope peptide of the present invention. ⁺ This is a method for quantifying cytokines and / or chemokines such as interferon gamma (IFNγ) and interleukin produced by CTL. The method is specifically described below using IFNγ as an example.
[0044]
(1) Method 1 by quantification of cytokines (quantification of intracellular IFNγ-producing cells);
Lymphocytes isolated from peripheral blood of a patient after HCT or HCT donor were placed in RPMI1640 medium containing 10% human serum at 2 × 10 5 ⁶ / Ml of the cells, and the mHA epitope peptide of the present invention is added at a concentration of 1 μg / ml. Further, Brefeldin A or the like, which is an inhibitor of intracellular protein transport, is added, and the cells are cultured at 37 ° C. for 5 to 6 hours in a carbon dioxide gas constant temperature bath. After the culture, the cells are fixed, subjected to membrane permeation treatment, and reacted with a dye-labeled anti-IFNγ antibody and an anti-CD8 antibody. CD8 using a flow cytometer ⁺ The% IFNγ-positive cells in the lymphocytes are quantified.
[0045]
(2) Method 2 by cytokine quantification (Elispot assay);
A 96-well MultiScreen-HA plate (Millipore) was coated with an anti-IFNγ monoclonal antibody overnight at 4 ° C., and each well was washed with PBS. Lymphocytes separated from peripheral blood of a patient after HCT or a HCT donor were collected from each well. Sprinkle. Put the mHA epitope peptide in each well and add 5% CO at 37 ° C. ₂ Incubate for 20 hours in an incubator. The next day, the plate is washed with PBS supplemented with 0.05% Tween-20, and then reacted with anti-IFNγ rabbit serum and peroxidase-labeled anti-rabbit IgG goat serum at room temperature for 90 minutes each. Further, 3-amino-9-ethylcarbasole (Sigma) and 0.015% H ₂ O ₂ And 0.1 M sodium acetate buffer (pH 5.0) is added to each well and reacted at room temperature for 40 minutes. IFNγ spots are visualized and counted with a stereomicroscope.
[0046]
(3) Method 3 by quantification of cytokines (method of quantifying IFNγ secreted into culture supernatant);
Peripheral blood of a post-HCT patient or HCT donor separated from peripheral blood was placed in RPMI1640 medium containing 10% human serum at 2 × 10 6 ⁶ MHA epitope peptide of the present invention at a concentration of 1 μg / ml. Incubate at 37 ° C for 24-48 hours in a carbon dioxide gas thermostat. After the culture, the supernatant is recovered, and the concentration of IFNγ contained therein is quantified using a commercially available ELISA kit (eg, HUMAN IFN gamma ELISA from ENDOGEN).
[0047]
As a second quantification method, MHC-tetramer prepared using the mHA epitope peptide of the present invention is used to recognize the mHA epitope peptide and to damage CD8 that can damage hematopoietic tumor cells or solid tumor cells. ⁺ CTL can be quantified. Preparation of the MHC-tetramer is as described above. The quantification can be performed, for example, as follows. Lymphocytes are separated from peripheral blood of a patient after HCT or an HCT donor and reacted with an appropriate concentration of MHC-tetramer at 37 ° C. for 15 minutes. CD8 bound to the tetramer ⁺ Since CTL is stained with a labeling dye, it is counted using a flow cytometer or the like.
[0048]
5. Compatibility genetic diagnosis
Combination useful for immunotherapy that enhances GVT by genetically diagnosing the compatibility between a donor and a patient in HCT therapy by performing a known PCR method using a region containing a polymorphic site of the bcl2A1 gene It can be diagnosed whether or not. The nucleotide sequence of at least one oligonucleotide primer and / or probe used in the PCR method includes a polymorphic site of the bcl2A1 gene, and as long as the polymorphic site possessed by the donor and the patient can be confirmed by the PCR method, The number of bases of the oligonucleotide primer and / or probe can be appropriately set within a commonly used range of 18 to 30 mer. 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.
[0049]
(1) Method by TaqMan PCR
PCR primers are designed to amplify the region flanking each polymorphic site of the bcl2A1 gene. In addition, a probe in which a different fluorescent substance (fluorescent reporter dye such as FAM or VIC) and a quencher are bound to a probe specific to the base sequence of the region containing each polymorphic site of the bcl2A1 gene is prepared, and together with the PCR primer When a PCR reaction is performed on sample DNA (DNA obtained by separating and purifying DNA from about 1 ml of peripheral blood of a patient and a donor using a commercially available DNA extraction kit (for example, QIAamp DNA Blood Mini Kit of Qiagen), etc.), Only when a base sequence is present, the corresponding probe is decomposed to emit fluorescence. In the PCR reaction, when there is one mismatch between the base sequence of the sample DNA and the base sequence of the probe, both sequences are hybridized under stringency conditions that do not hybridize. Stringency conditions vary depending on the hybridization conditions to be carried out, but those skilled in the art should appropriately set conditions such as temperature, salt concentration, solvent composition such as surfactant concentration, etc. based on the hybridization protocol. Can be. By measuring the generated fluorescence, the pattern of the 19th, 39th or 82nd amino acids of the bcl2A1 protein of the donor and the patient can be read. By performing typing between the donor and the patient, it is confirmed whether the mHA epitope peptide is compatible or incompatible, and if not, whether the combination is useful for immunotherapy for enhancing GVT. It is possible to diagnose whether or not.
[0050]
(2) Method using allele-specific PCR
The primer is designed so that one 3 ′ side of the PCR primer is specific to each nucleotide sequence of a region containing each polymorphic site of the bcl2A1 gene. The other primer is common to both alleles. When the PCR reaction is individually performed on the sample DNA prepared as described above, amplification occurs in the PCR reaction only when there is a primer compatible with the base sequence of the region containing each polymorphic site of the bcl2A1 gene, This makes it possible to determine the compatibility between a donor and a patient in HCT therapy.
[0051]
(3) Method using PCR-RFLP
PCR primers are designed to amplify a region containing each polymorphic site of the bcl2A1 gene. After performing a PCR reaction individually on the sample DNA prepared as described above, after digesting with a restriction enzyme that specifically recognizes the nucleotide sequence of the region containing each polymorphic site of each bcl2A1 gene Electrophoresis on a gel confirms the presence or absence of a region containing each polymorphic site of the bcl2A1 gene in the sample by the presence or absence of cleavage, and determines the compatibility between the donor and the patient in HCT therapy.
[0052]
(4) Direct nucleotide sequencing method
PCR primers are set so as to amplify a region containing each polymorphic site of the bcl2A1 gene, and a PCR reaction is individually performed on the sample DNA prepared as described above. The PCR amplification product is used as a template after purification, the nucleotide sequence is directly determined using the primers used for PCR, and the nucleotide sequence of the amplified portion of the region containing each polymorphic site of the bcl2A1 gene is directly read. Based on the results of the sequence, the compatibility between the donor and the patient in HCT therapy is determined.
[0053]
Based on the results of the various methods using PCR as described above, the donor and the polymorphic site of the bcl2A1 protein derived from the patient and / or the nucleotide sequence encoding the mHA epitope peptide of the bcl2A1 protein in the HCT therapy were confirmed by the donor in the HCT therapy. To determine whether the combination is useful for immunotherapy that enhances GVT. An example of the determination based on the result of genotyping the obtained PCR amplification product between the donor and the patient will be described below. However, it is not limited to this.
[0054]
The region containing the polymorphic site of the bcl2A1 gene, which is useful for confirming the compatibility of the bcl2A1 protein between the donor having the HLA-A24 and the patient and determining whether the immunotherapy for enhancing the GVT of the patient is effective includes: As an example, the mHA epitope peptide of SEQ ID NO: 3 and / or SEQ ID NO: 4 can be used.
[0055]
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 a base sequence encoding the mHA epitope peptide of SEQ ID NO: 3, and (2) DNA prepared from a patient. When a base sequence that hybridizes to the base sequence encoding the mHA epitope peptide of SEQ ID NO: 4 is present in the sample, both bcl2A1 proteins are incompatible. ⁺ CTLs can enhance a patient's GVT.
[0056]
Conversely, (1) a DNA sequence prepared from a donor contains a base sequence that hybridizes with a base sequence encoding the mHA epitope peptide of SEQ ID NO: 4, and (2) a DNA sample prepared from a patient contains: Even when a nucleotide sequence that hybridizes to the nucleotide sequence encoding the mHA epitope peptide of SEQ ID NO: 3 exists, both bcl2A1 proteins are incompatible and the donor-derived CD8 ⁺ CTLs can enhance a patient's GVT.
[0057]
Further, (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: 3 or 4 (that is, the donor is a homolog of the base sequence of SEQ ID NO: 3 or 4). (2) When the DNA sample prepared from the patient contains a nucleotide sequence that hybridizes with the nucleotide sequence encoding the mHA epitope peptide of SEQ ID NOS: 3 and 4 (that is, when the patient has SEQ ID NO: 3 and SEQ ID NO: 3). 4), the two bcl2A1 proteins are incompatible and the donor-derived CD8 ⁺ CTLs can enhance a patient's GVT.
[0058]
However, the DNA sample prepared from the donor and the DNA sample prepared from the patient may contain SEQ ID NO: 3 or 4 (ie, when homozygous for the nucleotide sequence of SEQ ID NO: 3 or 4) or 3 and 4 (ie, SEQ ID NO: 3 and 4). If the base sequence encoding the mHA epitope peptide having the same SEQ ID NO: exists in the case of heterozygous base sequence, the bcl2A1 protein is compatible and the donor-derived CD8 ⁺ CTL is not suitable for enhancing a patient's GVT.
[0059]
Further, a region containing a polymorphic site of the bcl2A1 gene, which is useful for confirming the compatibility of the bcl2A1 protein between a donor having HLA-B44 and a patient and determining whether immunotherapy for enhancing GVT of the patient is effective. As an example, the mHA epitope peptide of SEQ ID NO: 5 and / or SEQ ID NO: 6 can be used.
[0060]
For example, as a result of the various PCR methods, (1) a DNA sample prepared from a donor contains a sequence that hybridizes to a base sequence encoding the mHA epitope peptide of SEQ ID NO: 5, and (2) a DNA sample prepared from a patient. When a sequence that hybridizes with the nucleotide sequence encoding the mHA epitope peptide of SEQ ID NO: 6 is present, both bcl2A1 proteins are incompatible. ⁺ CTLs can enhance a patient's GVT.
[0061]
Conversely, (1) a DNA sample prepared from a donor contains a sequence that hybridizes to the nucleotide sequence encoding the mHA epitope peptide of SEQ ID NO: 6, and (2) a DNA sample prepared from a patient has SEQ ID NO: In the presence of a sequence that hybridizes with the nucleotide sequence encoding the mHA epitope peptide of No. 5, both bcl2A1 proteins are incompatible, and donor-derived CD8 ⁺ CTLs can enhance a patient's GVT.
[0062]
Furthermore, (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: 5 or 6 (that is, the donor is a homolog of the base sequence of SEQ ID NO: 5 or 6). (2) When the DNA sample prepared from the patient contains a nucleotide sequence that hybridizes with the nucleotide sequence encoding the mHA epitope peptide of SEQ ID NOS: 5 and 6 (that is, when the patient has SEQ ID NOs: 5 and 6). 6), both bcl2A1 proteins are incompatible and the donor-derived CD8 ⁺ CTLs can enhance a patient's GVT.
[0063]
However, when a DNA sample prepared from a donor and a DNA sample prepared from a patient contain a sequence that hybridizes with the nucleotide sequence encoding the mHA epitope peptide having the same SEQ ID NO: 5 or 6, or 5 or 6, Indicates that both bcl2A1 proteins are compatible and that CD8 ⁺ CTL is not suitable for enhancing a patient's GVT.
[0064]
In addition, among the nucleotide sequences encoding the peptides of these combinations, the PCR method can be carried out by using only the nucleotide sequence encoding any of the peptides. In order to surely confirm the above, it is desirable to use a nucleotide sequence encoding these peptides in combination.
[0065]
On the other hand, the opposite reaction of the GVT reaction is a host-graft reaction, which is a dangerous reaction that leads to rejection of the hematopoietic cells of the transfused donor during HCT. The peptide of the present invention may also be used for predicting the risk of rejection of transplanted hematopoietic cells, which is relatively likely to occur in patients with dysplastic syndrome.
[0066]
【Example】
Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.
[Example 1] CD8 capable of damaging hematopoietic tumor cells ⁺ Identification of CTL epitope peptide
1. Preparation of various materials
(1) Obtaining blood
Peripheral blood and HCT before and after transplantation of patients receiving HCT from HLA-identical siblings for leukemia (2 cases: acute myeloid leukemia (HLA-A24), chronic myelogenous leukemia (HLA-B44)) which are hematopoietic tumors Mononuclear cells were separated from donor peripheral blood. HLA type was measured using a serum method.
[0067]
(2) EBV infected B cell line
An HCT donor and a patient's peripheral blood mononuclear cells before transplantation are infected with the supernatant (including a live EBV virus) of an EBV-producing B95-8 cell line, and an EBV-infected B cell line (Lymphoblastoid cell line, hereinafter) , EBV-infected LCL).
In addition, peripheral blood mononuclear cells of healthy persons having HLA-A24 or HLA-B44 were similarly infected with the supernatant of B95-8 cell, which is an EBV-producing cell line, to establish EBV-infected LCL.
[0068]
2. CD8 specific for mHA epitope peptide ⁺ Establishment of CTL culture
(1) CD8 specific for mHA epitope peptide ⁺ CTL
1. The patient's peripheral blood mononuclear cells after HCT obtained in (1) were mixed and cultured in a flask together with the patient's peripheral blood mononuclear cells before HCT which had been irradiated with a lethal dose of radiation. There is an incompatibility of the mHA epitope peptide between the donor and the patient, and CD8 from donor lymphocytes in response to the patient's mHA epitope peptide (antigen) ⁺ CTLs began to proliferate. CD8 ⁺ To aid in the growth of CTL, IL-2 was added to the culture.
[0069]
(2) CD8 specific for the patient's mHA epitope peptide ⁺ Establishment of CTL clone and cytotoxicity test
2. Two CD8s obtained from two cases proliferated in (1) ⁺ CD8 from the CTL strain using limiting dilution ⁺ CTL clones were established. Each CD8 ⁺ Although a plurality of CTL clones were obtained from the CTL strain, the reaction patterns for EBV-infected LCLs prepared from healthy individuals were equivalent, and therefore, the clones were considered to be daughter clones originating from the same cells. Therefore, each CD8 ⁺ CTL clones having good growth were selected one by one from the CTL strain, and subjected to the following experiments. The two selected CTL clones are ⁵¹ In the cytotoxicity test using the Cr release method, the EBV-infected LCL of the HCT donor was not damaged, but the EBV-infected LCL of the patient was damaged, and thus it was considered to be specific for the mHA epitope peptide of the patient. In addition, from the reactivity to a plurality of EBV-infected LCLs prepared from healthy individuals having HLA-A24 or HLA-B44, two types of CD8 ⁺ The CTL clone was found to recognize the mHA epitope peptide presented by HLA-A24 and HLA-B44, respectively (hereinafter, HLA-A24 restricted CD8). ⁺ CTL clone, HLA-B44 restricted CD8 ⁺ CTL clone).
[0070]
Further, as shown in FIG. 7, four hematopoietic tumor patients (two acute myeloid leukemia FAB classification M2, one acute myeloid leukemia FAB classification M6, one chronic myelomonocytic leukemia CMMoL) ) Was collected from the first leukemia cells, and HLA-A24-restricted CD8 ⁺ When the cytotoxicity of the CTL clone was examined, not only EBV-infected LCL established from the same patient but also acute myeloid leukemia cells were strongly damaged, indicating that hematopoietic tumor cells were actually damaged. Although the CMMoL cells were not strongly damaged, the EBV-infected LCL established from the same patient was not strongly damaged, suggesting that the expression of the bcl2A1 gene was originally weak in this example.
[0071]
3. Identification of gene encoding mHA by linkage analysis
(1) Preparation of cell panel and cytotoxicity test
EBV-infected LCLs of CEPH (Centre d'Etude du Polymorphisme Humane) ancestry were obtained from the International Histocompatibility Workshop, and HLA-A24 and HLA-B44 genes were introduced into each of these EBV-infected LCLs to produce HLA-A24 or HLA. Each cell panel expressing -B44 on the cell surface was prepared. These are used as target cells; HLA-A24 restricted CD8 obtained in (2) ⁺ CTL clone and HLA-B44 restricted CD8 ⁺ Using CTL clone ⁵¹ A cytotoxicity test was performed using the Cr release method, and it was examined which EBV-infected LCL contained an mHA epitope peptide having the same polymorphism as that of a hematopoietic tumor patient. As a result, HLA-A24 restricted CD8 ⁺ CTL clone and HLA-B44 restricted CD8 ⁺ The CTL clone showed exactly the same pattern of cytotoxicity. HLA-A24 restricted CD8 ⁺ CTL clone and HLA-B44 restricted CD8 ⁺ FIG. 8 shows the results of the CTL clone.
[0072]
(2) Two-point linkage analysis
FASTLINK software and CEPH gene analysis database published on the Internet are available at ftp: // fastlink. nih. gov / pub / fastlink and http: // www. cephb. 2. obtained from fr / cephdb / Combination with the results of the cytotoxicity test obtained in (1), 2 HLA-A24 restricted CD8 obtained in (1) ⁺ CTL clone and HLA-B44 restricted CD8 ⁺ When the locus recognized by the CTL clone was identified, it was found that the long arm of chromosome 15 was around 24.3. Of the 32 genes at this locus, only bcl2A1 has two or more polymorphic sites and can encode multiple mHA epitope peptides presented on both HLA-A24 and HLA-B44. Met.
[0073]
4. Identification of mHA epitope peptide capable of binding to HLA-A2 or HLA-B44
Regarding the amino acid sequence of the bcl2A1 protein shown in SEQ ID NO: 1, an epitope peptide consisting of 9 to 10 amino acids having a binding motif of HLA-A24 or HLA-B44 can be searched, and BioInformatics & Molecular Analysis published on the Internet can be searched. against the HLA Peptide binding Predictions of Section (BIMAS) (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html.), 9~10 having a binding motif for HLA-A24 or HLA-B44 A large number of mHA epitope candidate peptides consisting of 10 amino acids were screened, and six types of mHA epitope candidate peptides were synthesized. .
this ⁵¹ After the cells were added to mHA-negative HCT donor cells that had been radiolabeled with Cr and allowed to stand at room temperature for 30 minutes, an appropriate number of 2. HLA-A24 restricted CD8 obtained in (2) ⁺ CTL clone or HLA-B44 restricted CD8 ⁺ After mixing with the CTL clone, a cytotoxicity test was performed.
[0074]
Also, CD8 ⁺ In order to confirm that the CTL mHA epitope peptide is processed endogenously, a minigene encoding each polymorphic site of the bcl2A1 gene was prepared, introduced into an EBV-infected LCL of a mHA-negative donor, and presented with antigen. Cells ⁵¹ The Cr release test was performed similarly. Here, the term “endogenously processed” means that bcl2A1 protein newly produced in the mini-gene-transfected cell is cleaved into an antigen peptide of a correct length by an antigen processing mechanism inherent in the cell, and the HLA heavy chain is cleaved. It means a state that forms a complex with β2 microglobulin and is displayed on the surface of bcl2A1 minigene-introduced cells.
[0075]
CD8 according to the above test method ⁺ CTL and its CD8 ⁺ MHA epitope peptides represented by the following SEQ ID NOs: 3 to 8 to which the CTL clone had reacted were obtained. Among these peptides, SEQ ID NOS: 3 and 4 are peptide sequences having an HLA-A24 binding motif, and SEQ ID NOs: 5 to 8 are peptide sequences having an HLA-B44 binding motif.
[0076]
The mHA epitope peptides of SEQ ID NOS: 3 and 4 are partial peptides comprising the 19th amino acid in the amino acid sequence of the bcl2A1 protein represented by SEQ ID NO: 1, and the mHA epitope peptides of SEQ ID NOs: 5 to 8 are It is a partial peptide comprising the 82nd amino acid in the amino acid sequence represented by SEQ ID NO: 1.
Asp Tyr Leu Gln Cys Val Leu Gln Ile (SEQ ID NO: 3)
Asp Tyr Leu Gln Tyr Val Leu Gln Ile (SEQ ID NO: 4)
Lys Glu Phe Glu Asp Asp Ile Ile Asn Trp (SEQ ID NO: 5)
Lys Glu Phe Glu Asp Gly Ile Ile Asn Trp (SEQ ID NO: 6)
Glu Phe Glu Asp Asp Ile Ile Asn Trp (SEQ ID NO: 7)
Glu Phe Glu Asp Gly Ile Ile Asn Trp (SEQ ID NO: 8)
[0077]
[Example 2] Identification of mHA epitope peptide capable of binding to HLA-A2
The peptides represented by SEQ ID NOs: 3 to 8 are partial peptides containing the 19th amino acid or the 82nd amino acid, respectively, of the bcl2A1 protein, and also bind to HLA-A24 or HLA-B44. Although it was a peptide, in order to further examine an mHA epitope peptide capable of binding to HLA-A2, the amino acid sequence of the bcl2A1 protein was analyzed on the HLA Peptide of BioInformatics & Molecular Analysis Section (BIMAS) published on the Internet. As a result of matching by Predictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html.), H It was found that an mHA epitope consisting of 9 to 10 amino acids having an LA-A2 binding motif was present, and the following eight epitope candidate peptides were identified.
[0078]
Among these sequences, the peptides represented by SEQ ID NOS: 9 to 12 are partial peptides containing the 19th amino acid in the amino acid sequence of the bcl2A1 protein, and the peptides represented by SEQ ID NOs: 13 to 16 correspond to the bcl2A1 protein. Is a partial peptide containing the 39th amino acid.
Arg Leu Ala Gln Asp Tyr Leu Gln Cys (SEQ ID NO: 9)
Arg Leu Ala Gln Asp Tyr Leu Gln Tyr (SEQ ID NO: 10)
Leu Ala Gln Asp Tyr Leu Gln Cys Val (SEQ ID NO: 11)
Leu Ala Gln Asp Tyr Leu Gln Tyr Val (SEQ ID NO: 12)
Lys Thr Ser Arg Val Leu Gln Asn Val (SEQ ID NO: 13)
Lys Thr Ser Arg Val Leu Gln Lys Val (SEQ ID NO: 14)
Val Leu Gln Asn Val Ala Phe Ser Val (SEQ ID NO: 15)
Val Leu Gln Lys Val Ala Phe Ser Val (SEQ ID NO: 16)
[0079]
[Example 3] Vaccine injection
Each of the peptides of SEQ ID NOs: 3 to 16 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 sterile vials 1 ml at a time to obtain a vaccine injection.
[0080]
[Example 4] CD8 that damages hematopoietic tumor cells or solid tumor cells in peripheral blood of a patient after HCT using MHC-tetramer prepared from mHA epitope peptide ⁺ Method for quantifying CTL
1. Preparation of MHC-tetramer
(1) Preparation of MHC
HLA A * 2402 heavy chain and β2 microglobulin were prepared and purified in large quantities using a recombinant protein expression system using Escherichia coli. Note that an amino acid sequence recognized by biotin ligase was added to the C-terminus of HLA A * 2402 heavy chain in advance. Purified HLA A * 2402 heavy chain and β2 microglobulin were each dissolved in 8M urea. 200 ml of rebuffering buffer (pH 8.0; 100 mM Tris-HCl, 400 mM L-arginine-HCl, 2 mM EDTA, 0.5 mM oxidative glutathione, 5 mM reducedglutinite in 5 mM reduced glueone in peptide sequence, 5 mM reduced glutaion) 18.6 mg of HLA A * 2402 heavy chain and 13.2 mg of β2 microglobulin were each injected using a syringe with a 27 gauge needle. After stirring in a 10 ° C. constant temperature bath for 48 to 72 hours to promote 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 refolding after dialysis was performed. The buffer was concentrated to 2 ml using Centriprep 10 (MILLIPORE, Bedford, MA). MHC flowing out at a molecular weight of about 45 KD was isolated by gel filtration chromatography using a Superdex 200 HR (Amersham Pharmacia Biotech AB, Uppsala, Sweden) column.
[0081]
(2) Preparation of biotinylated MHC
Biotin was bound to a specific site at the C-terminus of the HLA A * 2402 heavy chain using biotin ligase (AVIDITY, Denver, CO). Biotin-added MHC was purified by gel filtration chromatography using a Superdex 200 HR column.
[0082]
(3) Preparation of MHC-tetramer
PE-labeled streptavidin (Molecular Probe, Eugene, OR) and purified biotinylated MHC were mixed at a molar ratio of 1: 4. An MHC-tetramer containing the peptide of SEQ ID NO: 4 flowing out around a molecular weight of 480 KD was isolated by gel filtration chromatography using a Superdex 200 HR column. It was concentrated to about 3 mg / ml using Centricon 10 (MILLIPORE) and stored at 4 ° C. As preservatives, Na-Azide, EDTA, Leupeptin and Pepstatin were added.
[0083]
2. CD8 damaging hematopoietic tumor cells or solid tumor cells using MHC-tetramer ⁺ CTL quantification
Peripheral blood lymphocytes (1 × 10 4) isolated from a patient with HLA-A24 that underwent HCT with an incompatible mLA epitope peptide presented at the HLA-A24 of the bcl2A1 protein at 43 days post-HCT ⁶ ) Or lymphocytes proliferating after stimulation of peripheral blood lymphocytes of the patient with the peptide shown in SEQ ID NO: 2 for 2 weeks (1 × 10 ⁶ ) Was suspended in 50 μL of PBS containing 2% FCS in a 1.5 ml Eppendorf tube. To this, MHC-tetramer containing the peptide of SEQ ID NO: 4 and FITC-labeled anti-CD8 antibody (CALTAG, Burlingame, Calif.) Were added in an amount of 1 μL each, and the mixture was allowed to stand in a thermostat at 37 ° C. for 15 minutes to react. The cells after the reaction were washed three times with 1 ml of 2% FCS-containing PBS, and then suspended in 0.5% paraformaldehyde-containing PBS (1 ml). CD8 bound to the MHC-tetramer ⁺ CTLs are stained with a labeling dye and FITC-CD8 using FACS Calibur (Becton Dickinson). ⁺ Tetramer positive cells in the T cells were counted and the ratio was calculated.
[0084]
The result of the analysis is shown in FIG. CD8 bound to MHC-tetramer and damaging hematopoietic or solid tumor cells ⁺ CTL moves to the right as it is stained. In the figure, A is a sample obtained by staining peripheral blood of an HCT donor (negative control), and B is CD8 binding to the peptide of SEQ ID NO: 4. ⁺ It is a CTL clone (positive control). C is peripheral blood 43 days after transplantation of a patient who has received a mismatched HCT of the mHA epitope peptide of the bcl2A1 protein (population in the upper right corner of FIG. C is tetramer positive cells), and D is the peptide of SEQ ID NO: 4 These are lymphocytes that have proliferated after stimulating the peripheral blood of the patient used twice for two weeks. These results indicate that the MHC-tetramer was stimulated with the mHA epitope peptide of SEQ ID NO: 4 and proliferated CD8. ⁺ It is clear that CTL is specifically stained.
[0085]
【The invention's effect】
According to the present invention, it is a partial peptide comprising 9 to 10 amino acid residues of the bcl2A1 protein, and binds to human major histocompatibility antigen (HLA) to form CD8. ⁺ An mHA epitope peptide that can be recognized by CTL can be provided. In addition, the peptide can be used to treat or prevent hematopoietic tumors or solid tumors in patients who have undergone HCT. Furthermore, CD8 that damages hematopoietic tumor cells or solid tumor cells in the patient after HCT ⁺ CTL can be quantified. It is also possible to provide a method for diagnosing suitability for allogeneic hematopoietic cell transplantation using the peptide.
[0086]
[Sequence list]

[Brief description of the drawings]
FIG. 1 CD8 ⁺ It is a figure which shows the recognition mechanism of hematopoietic tumor cells or solid tumor cells by CTL.
FIG. 2 shows a method for preparing an MHC-tetramer.
FIG. 3. MHC-tetramer and CD8 ⁺ It is a figure which shows the binding of CTL.
FIG. 4 is a diagram showing a method for preparing MHC-magnetic beads.
FIG. 5: CD8 by MHC-magnetic beads ⁺ FIG. 4 shows the isolation of CTL.
FIG. 6: CD8 upon stimulation with mHA epitope peptide and subsequent anti-CD3 antibody, IL-2 ⁺ It is a figure which shows the proliferation of CTL.
FIG. 7: CD8 binding to HLA-A24 restricted clone ⁺ It is a figure which shows the cytotoxicity of various leukemia cells by CTL.
[FIG. 8] HLA-A24- and HLA-B44-restricted clones have exactly the same pattern as those obtained by expressing HLA-A24 and HLA-B44 in EBV-infected LCLs established from CEPH kindreds, respectively. It is a figure showing that it is injured.
FIG. 9 shows CD8 that damages hematopoietic tumor cells or solid tumor cells by a tetramer prepared with the peptide of SEQ ID NO: 4 of the present invention. ⁺ It is a figure which shows identification and quantification of CTL.

Claims (19)

a partial histocompatibility peptide consisting of 9 to 10 amino acid residues of the bcl2A1 protein and capable of binding to human major histocompatibility antigen (HLA) and being recognized by CD8 ⁺ cytotoxic T lymphocytes Antigen (mHA) epitope peptide. The partial peptide consisting of 9 to 10 amino acid residues of the bcl2A1 protein is a partial peptide comprising the 19th, 39th or 82nd amino acids in the amino acid sequence represented by SEQ ID NO: 1. The peptide according to claim 1. The peptide according to claim 1 or 2, wherein the HLA is HLA-A24. The peptide according to claim 3, which is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. The peptide according to claim 1 or 2, wherein the HLA is HLA-B44. The peptide according to claim 5, which is a peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 5 to 8. The peptide according to claim 1 or 2, wherein the HLA is HLA-A2. The peptide according to claim 7, which is a peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 9 to 16. The peptide according to any one of claims 1 to 8, wherein the CD8 ⁺ cytotoxic T lymphocyte damages hematopoietic tumor cells or solid tumor cells. The peptide according to claim 9, wherein the hematopoietic tumor is leukemia, myelodysplastic syndrome, malignant lymphoma or multiple myeloma. 10. The peptide according to claim 9, wherein the solid tumor is renal cell carcinoma, malignant melanoma, breast cancer, gastric cancer, lung cancer or prostate cancer. The peptide according to any one of claims 1 to 11 or an antigen-presenting cell pulsed with the peptide as an active ingredient, for treating a hematopoietic tumor or a solid tumor that has remained or recurred after allogeneic hematopoietic cell transplantation, or A vaccine for preventing recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation. 12. Allogeneic hematopoietic cell transplantation comprising a peptide according to any one of claims 1 to 11 or a CD8 ⁺ cytotoxic T lymphocyte obtained by stimulating peripheral blood lymphocytes with antigen-presenting cells pulsed with the peptide. A passive immunotherapy agent for treating a residual or recurrent hematopoietic tumor or a solid tumor, or for preventing a recurrence of a hematopoietic tumor or a solid tumor after allogeneic hematopoietic cell transplantation. A major histocompatibility antigen by reacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the peptide according to any one of claims 1 to 11 with peripheral blood lymphocytes. complexes 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 a hematopoietic tumor or a solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation, or for preventing a recurrence of a 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 11 is reacted with peripheral blood lymphocytes, and the major histocompatibility complex-labeled magnetic bead is reacted with CD8 ^+. A hematopoietic tumor or solid tumor remaining or recurring after allogeneic hematopoietic cell transplantation, comprising a conjugate to which cytotoxic T lymphocytes are bound and containing CD8 ⁺ cytotoxic T lymphocytes obtained by isolation from the conjugate Or a passive immunotherapeutic agent for preventing recurrence of hematopoietic tumors or solid tumors after allogeneic hematopoietic cell transplantation. A peripheral blood is stimulated with the peptide according to any one of claims 1 to 11 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 damages hematopoietic tumor cells or solid tumor cells, which comprises measuring. A major histocompatibility complex and / or a major histocompatibility complex-tetramer is prepared from the peptide according to any one of claims 1 to 11, and the major histocompatibility complex and / or major histocompatibility is prepared. A method for quantifying CD8 ⁺ cytotoxic T lymphocytes that damage hematopoietic tumor cells or solid tumor cells in peripheral blood, by reacting the complex-tetramer with peripheral blood. DNA from the donor and the patient in the allogeneic hematopoietic cell transplantation was amplified by PCR using the nucleotide sequence of the region containing the polymorphic site of the bcl2A1 gene shown in SEQ ID NO: 2, and the obtained PCR amplification product was A method for diagnosing suitability for allogeneic hematopoietic cell transplantation, comprising performing genotyping. 19. The diagnostic method according to claim 18, wherein the nucleotide sequence of the region containing the polymorphic site of the bcl2A1 gene shown in SEQ ID NO: 2 is a nucleotide sequence encoding the mHA epitope peptide according to claim 1.

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